Agonist antibodies that bind human CD137 and uses thereof

ABSTRACT

The present disclosure relates to compounds (e.g., antibodies, or antigen-binding fragments thereof) that bind to an epitope of CD137 and agonize CD137, and to use of the compounds in methods for treating, or ameliorating one or more symptoms of, cancer.

RELATED APPLICATIONS

This application is a Divisional of Continuation patent application Ser.No. 16/123,742, filed Sep. 6, 2018, now pending, which application is aContinuation of U.S. patent application Ser. No. 16/032,639, filed Jul.11, 2018, Abandoned, which application claims the benefit of U.S.Provisional Patent Application Ser. No. 62/531,259 filed on Jul. 11,2017; U.S. Provisional Patent Application Ser. No. 62/531,190 filed onJul. 11, 2017; U.S. Provisional Patent Application Ser. No. 62/568,231filed on Oct. 4, 2017; U.S. Provisional Patent Application Ser. No.62/577,257 filed on Oct. 26, 2017; and U.S. Provisional PatentApplication Ser. No. 62/577,259 filed on Oct. 26, 2017. The entirecontents of the above-referenced applications are incorporated herein bythis reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Dec. 12, 2018, isnamed “CTN-006CNDV_Sequence-Listing.txt” and is 92674 Kilobytes in size.

BACKGROUND

In recent years, an increasing body of evidence suggests the immunesystem operates as a significant barrier to tumor formation andprogression. The principle that naturally-occurring T cells withanti-tumor potential or activity exist in a patient with cancer hasrationalized the development of immunotherapeutic approaches inoncology. Immune cells, such as T cells, macrophages, and natural killercells, can exhibit anti-tumor activity and effectively control theoccurrence and growth of malignant tumors. Tumor-specific or -associatedantigens can induce immune cells to recognize and eliminate malignancies(Chen & Mellman, (2013) Immunity 39(1):1-10). In spite of the existenceof tumor-specific immune responses, malignant tumors often evade oravoid immune attack through a variety of immunomodulatory mechanismsresulting in the failure to control tumor occurrence and progression(Motz & Coukos, (2013) Immunity 39(1):61-730). Indeed, an emerginghallmark of cancer is the exploitation of these immunomodulatorymechanisms and the disablement of anti-tumor immune responses, resultingin tumor evasion and escape from immunological killing (Hanahan andWeinberg (2011) Cell 144(5):646-674).

Novel approaches in the immunotherapy of cancer involve counteractingthese immune evasion and escape mechanisms and inducing the endogenousimmune system to reject tumors. CD137 (alternatively known as “tumornecrosis factor receptor superfamily member 9” (TNFRSF9), 4-1BB, and“induced by lymphocyte activation” (ILA)) is a transmembraneco-stimulatory receptor protein belonging to the tumor necrosis factorsuperfamily. CD137 is a T cell co-stimulatory receptor induced upon TCRactivation (Nam et al., (2005) Curr Cancer Drug Targets 5:357-363; Wattset al., (2005) Annu Rev Immunol 23:23-68). In addition to its expressionon activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+regulatory T cells, activated natural killer (NK) and NK-T cells,monocytes, neutrophils, and dendritic cells.

Under physiological conditions, CD137 is ligated by CD137 ligand(CD137L), an agonist membrane molecule present on antigen-presentingcells including B cells, monocytes, macrophages, and dendritic cells(Watts et al., (2005) Annu Rev Immunol 23:23-68). Upon interaction withits ligand, CD137 leads to increased TCR-induced T-cell proliferation,cytokine production, functional maturation, and prolonged CD8+ T-cellsurvival. The potential of CD137 co-stimulation using various agonists(e.g. agonistic antibodies, recombinant CD137L protein, andCD137-specific aptamers) in enabling the immune system to attack tumorshas been documented in numerous models (Dharmadhikari et al., (2016)Oncoimmunology 5(4): e1113367 and references therein). A recent reporton the clinical evaluation of an agonistic CD137 antibody (Urelumab,BMS-663513; Bristol-Myers Squibb) documented the observation oftreatment-related adverse events in human subjects, includingindications of severe hepatotoxicity (transaminitis) correlating withantibody dose (Segal et al., (2016) Clin Cancer Res 23(8):1929-1936). Incontrast, a different agonistic CD137 antibody (Utomilumab, PF-05082566;Pfizer) tested in combination with an anti-PD-1 antibody(pembrolizumab), though not resulting in any dose-limiting toxicities,showed comparable results to anti-PD-1 antibody therapy alone (Tolcher,A. et al., (2017) Clin Cancer Res 23(18): 5349-5357). These resultshighlight that for patients with various diseases and conditions,including cancer, that are amenable to treatment with a CD137 agonist,there continues to be an unmet need for novel agonistic antibodies thatbind to human CD137 and exhibit characteristics sufficient for thedevelopment of a safe and efficacious therapeutic.

SUMMARY OF THE DISCLOSURE

The present disclosure is based, at least in part, on the discovery ofnovel agonist anti-CD137 antibodies exhibiting protective anti-tumorimmunity in animals. Notably, the antibodies described herein areefficacious against diverse tumor types, and over a wide dose range.Moreover, as exemplified in the working examples, the antibodiesdescribed herein are therapeutically effective against very largetumors. For example, treatment of tumor-bearing mice with agonistanti-CD137 antibodies described herein resulted in complete regressionof tumors as large as 1,800 mm³. As set forth in FIG. 15, treatment ofsuch mice also resulted in protective immunity. And coincident with theobserved efficacy were positive immunophenotypic changes in the tumormicroenvironment, such as increased immune cell infiltration withconcomitant reductions in regulatory T cell and exhausted T cellpopulations (see, e.g., FIGS. 22A-22D).

As described above, agonism of CD137 has been associated with certainadverse events, including hepatotoxicity-related deaths in humans (see,e.g., Segal et al. (2017) Clin Cancer Res 23(8): 1929-1935). Similartoxicities resulting from treatment with agonist anti-CD137 antibodies(such as the 3H3 antibody) have also been observed in animal models(see, e.g., Bartkowiak et al. (2018) Clin Cancer Res 24(5):1138-1151).Yet, the agonist anti-CD137 antibodies described herein have minimaleffects on the liver, as determined by, e.g., plasma levels of liverenzymes (e.g., alanine aminotransferase (ALT)) and immune cellinfiltration. For example, there was no evidence of increasedintrahepatic or intrasplenic immune cell infiltration in mice treatedwith the antibodies. Thus, the antibodies described herein are not onlyhighly efficacious, but also sparing of certain toxicities associatedwith CD137 agonism.

While the disclosure is not bound by any particular theory or mechanismof action, the superior therapeutic and toxicity-sparing properties ofthe antibodies described herein are believed to derive in part from oneor both of their affinity and the novel epitope to which they bind. Thatis, the antibodies described herein share a common, novel epitope thatis distinct from that of other agonist anti-CD137 antibodies. And, asexemplified in the working examples, engagement of this epitope by theantibodies described herein gives rise to differentiated in vitroactivity, such as effects on regulatory T cell proliferation, cytokineproduction by CD8⁺ T cells and macrophages, and intracellular signaling,as compared to agonist antibodies that bind to different epitopes ofCD137. Furthermore, it has been demonstrated that an affinity range (a“sweet spot”) for antibodies is particularly optimal for anti-tumoractivity. For example, antibodies of intermediate affinity were shown tobe more efficacious against large tumors as compared to antibodies withhigher or lower affinity.

In view of the foregoing, in some aspects, the disclosure providesisolated monoclonal antibody, or antigen binding portion thereof, thatspecifically binds human CD137, wherein the antibody or antigen bindingportion binds human CD137 with an affinity (K_(D)) of between about 40nM to about 100 nM. In some aspects, the disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof, thatspecifically binds to human CD137, wherein the antibody or antigenbinding portion binds human CD137 with an affinity (K_(D)) of about30-100 nM (e.g., between about 30 nM and about 110 nM). In some aspects,the affinity of the anti-CD137 antibody to human CD137 is at least two(e.g., at least three, four, five, six, seven, eight, nine, or 10) foldhigher than the affinity of mAb10 for mouse CD137. In some aspects, theaffinity of the anti-CD137 antibody is no greater than 500, 450, 400,350, 300, 250, 200, 250, 200, 175, 150, 125, 110, or 100 nM. In someaspects, the affinity of the anti-CD137 antibody to human CD137 is atleast two (e.g., at least three, four, five, six, seven, eight, nine, or10) fold higher than the affinity of mAb10 for mouse CD137, but nogreater than 500, 450, 400, 350, 300, 250, 200, 250, 200, 175, 150, 125,110, or 100 nM.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein the antibody or antigen binding portion binds to anepitope on human CD137 comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or all 25) of amino acids 111-132 of SEQ IDNO:3. In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein the antibody or antigen binding portion binds to anepitope within amino acids 111-132 of SEQ ID NO:3. In some aspects, thedisclosure provides an isolated monoclonal antibody, or antigen bindingportion thereof, that specifically binds to human CD137, wherein theantibody or antigen binding portion binds to all or a portion of aminoacids 111-132 of SEQ ID NO:3. In some aspects, the epitope comprisesK114 of SEQ ID NO: 3. In some aspects, the epitope comprises residuesE111, T113, and K114 of SEQ ID NO: 3. In some aspects, the epitopecomprises residues E111, T113, K114, N126 and I132 of SEQ ID NO: 3. Insome aspects, the epitope comprises residues E111, T113, K114 and P135of SEQ ID NO: 3. In some aspects, the epitope comprises residues E111,T113, K114, N126, I132 and P135 of SEQ ID NO: 3. In some aspects, theantibody or antigen binding portion thereof binds to human CD137 with anaffinity of between about 30 nM and about 100 nM (e.g., between about 30nM and about 110 nM).

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein the antibody or antigen binding portion binds humanCD137 with an affinity (K_(D)) of about 40-100 nM (e.g., between about40 nM and about 100 nM) and binds to an epitope on human CD137comprising K114 of SEQ ID NO: 3. In some aspects, the disclosureprovides an isolated monoclonal antibody, or antigen binding portionthereof, that specifically binds to human CD137, wherein the antibody orantigen binding portion binds human CD137 with an affinity (K_(D)) ofabout 30-100 nM (e.g., between about 30 nM and about 100 nM) and bindsto an epitope on human CD137 comprising K114 of SEQ ID NO: 3. In someaspects, the epitope comprises residues E111, T113, and K114 of SEQ IDNO: 3. In some aspects, the epitope comprises residues E111, T113, K114,N126 and I132 of SEQ ID NO: 3. In some aspects, the epitope comprisesresidues E111, T113, K114 and P135 of SEQ ID NO: 3. In some aspects, theepitope comprises residues E111, T113, K114, N126, I132 and P135 of SEQID NO: 3.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein the antibody or antigen binding portion binds humanCD137 with an affinity (K_(D)) of about 30-100 nM (e.g., about 30 nM toabout 100 nM) and binds to an epitope on human CD137 comprising asequence of one or more amino acid residues corresponding to amino acidpositions 111 to 135 of SEQ ID NO: 3. In some aspects, the epitopecomprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 amino acid residues corresponding to aminoacid positions 111 to 135 of SEQ ID NO: 3.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein the antibody or antigen binding portion binds humanCD137 with an affinity (K_(D)) of about 30-100 nM (e.g., between about30 nM and about 100 nM) and binds to an epitope on human CD137 locatedwithin amino acid residues 111-135 of SEQ ID NO: 3. In some aspects, theepitope is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 aminoacids. In some aspects, the epitope is fewer than 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein the antibody or antigen binding portion binds humanCD137 with an affinity (K_(D)) of about 30-100 nM (e.g., between about30 nM and about 100 nM) and binds to an epitope on human CD137comprising ELTK (corresponding to amino acid residues 111-114 of SEQ IDNO: 3). In some aspects, the epitope further comprises one or moreresidues N126, I132 and P135 of SEQ ID NO: 3.

In any of the foregoing aspects, the epitope is a non-linear epitope. Inany of the foregoing aspects, mutation of residue K114 of SEQ ID NO: 3abrogates binding of the antibody or antigen binding portion thereof tohuman CD137.

In any of the foregoing aspects, the antibody or antigen binding portiondescribed herein binds human CD137 with an affinity (K_(D)) of about30-100 nM, 30-95 nM, 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM,55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM. In some aspects, the antibodyor antigen binding portion binds to a non-ligand binding region of theextracellular domain of human CD137. In some aspects, the antibody orantigen binding portion does not inhibit the interaction between CD137and CD137L. In some aspects, the non-ligand binding region spanscysteine rich domain (CRD) III and CRD IV. In any of the foregoingaspects, the antibody or antigen binding portion does not inhibit theformation of a trimer of CD137:CD137L monomers.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion:

(i) binds human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) binds to a non-ligand binding region of the extracellular domain ofhuman CD137; and

(iii) binds to an epitope on human CD137 comprising K114 of SEQ ID NO:3.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion:

(i) binds human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) does not inhibit the interaction between human CD137 and humanCD137 ligand; and

(iii) binds to an epitope on human CD137 comprising K114 of SEQ ID NO:3.

In some aspects, the disclosure features an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion: (i) bindshuman CD137 with an affinity (K_(D)) of about 30-100 nM (e.g., betweenabout 30 nM and about 100 nM) and (ii) does not inhibit the formation ofa trimer of CD137:CD137L monomers (that is, a CD137:CD137L trimer:trimercomplex). In some aspects, the disclosure features an isolatedmonoclonal antibody, or antigen binding portion thereof, thatspecifically binds human CD137, wherein the antibody or antigen bindingportion: (i) binds human CD137 with an affinity (K_(D)) of about 30-100nM (e.g., between about 30 nM and about 100 nM) and (ii) binds to anon-ligand binding region of the extracellular domain of human CD137. Insome aspects, the disclosure features an isolated monoclonal antibody,or antigen binding portion thereof, that specifically binds human CD137,wherein the antibody or antigen binding portion: (i) binds human CD137with an affinity (K_(D)) of about 30-100 nM (e.g., between about 30 nMand about 100 nM) and (ii) does not inhibit the interaction betweenhuman CD137 and CD137 ligand.

In any of the foregoing aspects, the antibody or antigen binding portioncomprises a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid. In someaspects, the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXPFXLDXXYYYYYX (SEQ ID NO:127), wherein X is any amino acid. In any of the foregoing aspects,mutation of residues D95, L100, Y100E, Y100G, Y100H, or combinationsthereof, of the heavy chain CDR3, to alanine results in loss of bindingto human CD137. In any of the foregoing aspects, mutation of residuesP97, F98, D100A, Y100D, Y100F, or combinations thereof, to alanineresults in reduction of binding to human CD137. In any of the foregoingaspects, the antibody or antigen binding portion comprises heavy andlight chain CDRs, wherein heavy chain CDR3 comprises the amino acidsequence set forth in SEQ ID NO: 68.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM); and

(ii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid. In some aspects, X is any amino acidexcept alanine.

In another aspect, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM); and

(ii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X7X₈YX₉YYX₁₀(SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid. Insome aspects, X₂ is proline, X₃ is phenylalanine or tryptophan, X₅ isaspartic acid or glutamic acid, X₈ is tyrosine, and X₉ is tyrosine.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM); and

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope on human CD137 comprising one or more residues E111, T113,K114, N126, I132 and P135 of SEQ ID NO: 3.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope on human CD137 comprising one or more residues E111, T113,K114, N126, I132 and P135 of SEQ ID NO: 3;

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid; or

(iv) combinations thereof. In some aspects, X is any amino acid exceptalanine.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope on human CD137 comprising one or more residues E111, T113,K114, N126, I132 and P135 of SEQ ID NO: 3;

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀(SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid; or

(iv) combinations thereof. In some aspects, X₂ is proline, X₃ isphenylalanine or tryptophan, X₅ is aspartic acid or glutamic acid, X₈ istyrosine, and X₉ is tyrosine.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope on human CD137 comprising one or more residues E111, T113,K114, N126, I132 and P135 of SEQ ID NO: 3; and

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid. In some aspects, X is any amino acidexcept alanine.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope on human CD137 comprising one or more residues E111, T113,K114, N126, I132 and P135 of SEQ ID NO: 3; and

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀(SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid. Insome aspects, X₂ is proline, X₃ is phenylalanine or tryptophan, X₅ isaspartic acid or glutamic acid, X₈ is tyrosine, and X₉ is tyrosine.

In any of the foregoing aspects, the epitope comprises K114. In any ofthe foregoing aspects, the epitope comprises E111, T113 and K114. In anyof the foregoing aspects, the epitope comprises E11, T113, K114, N126and I132. In any of the foregoing aspects, the epitope comprisesresidues E111, T113, K114, N126, I132 and P135 of SEQ ID NO: 3.

In another aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM); and

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising a sequence of one or more amino acid residuescorresponding to amino acid positions 111 to 135 of SEQ ID NO: 3.

In another aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising a sequence of one or more amino acid residuescorresponding to amino acid positions 111 to 135 of SEQ ID NO: 3;

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid; or

(iv) combinations thereof. In some aspects, X is any amino acid exceptalanine.

In another aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising a sequence of one or more amino acid residuescorresponding to amino acid positions 111 to 135 of SEQ ID NO: 3;

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀(SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid; or

(iv) combinations thereof. In some aspects, X₂ is proline, X₃ isphenylalanine or tryptophan, X₅ is aspartic acid or glutamic acid, X₈ istyrosine, and X₉ is tyrosine.

In another aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising a sequence of one or more amino acid residuescorresponding to amino acid positions 111 to 135 of SEQ ID NO: 3; and

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid. In some aspects, X is any amino acidexcept alanine.

In another aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising a sequence of one or more amino acid residuescorresponding to amino acid positions 111 to 135 of SEQ ID NO: 3; and

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀(SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid. Insome aspects, X₂ is proline, X₃ is phenylalanine or tryptophan, X₅ isaspartic acid or glutamic acid, X₈ is tyrosine, and X₉ is tyrosine.

In any of the foregoing aspects, the epitope comprises 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25amino acid residues corresponding to amino acid positions 111 to 135 ofSEQ ID NO: 3.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity of about 30-100 nM (e.g., between about 30 nM and about 100nM); and

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising ELTK (corresponding to amino acid residues111-114 of SEQ ID NO: 3).

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising ELTK (corresponding to amino acid residues111-114 of SEQ ID NO: 3);

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid; or

(iv) combinations thereof. In some aspects, X is any amino acid exceptalanine.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising ELTK (corresponding to amino acid residues111-114 of SEQ ID NO: 3);

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀(SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid; or

(iv) combinations thereof. In some aspects, X₂ is proline, X₃ isphenylalanine or tryptophan, X₅ is aspartic acid or glutamic acid, X₈ istyrosine, and X₉ is tyrosine.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising ELTK (corresponding to amino acid residues111-114 of SEQ ID NO: 3); and

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid. In some aspects, X is any amino acidexcept alanine.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically binds tohuman CD137, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM);

(ii) the antibody or antigen binding portion thereof specifically bindsto an epitope comprising ELTK (corresponding to amino acid residues111-114 of SEQ ID NO: 3); and

(iii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid. Insome aspects, X₂ is proline, X₃ is phenylalanine or tryptophan, X₅ isaspartic acid or glutamic acid, X₈ is tyrosine, and X₉ is tyrosine

In any of the foregoing aspects, the epitope comprises the residues ELTKof SEQ ID NO: 3 (corresponding to amino acid residues 111-114 of SEQ IDNO: 3). In some aspects, the epitope comprises ELTK of SEQ ID NO: 3(corresponding to amino acid residues 111-114 of SEQ ID NO: 3) andresidues N126, I132 and P135 of SEQ ID NO: 3.

In any of the foregoing aspects, the epitope is a non-linear epitope. Insome aspects, mutation of residue K114 of human CD137 (SEQ ID NO: 3)abrogates binding of the antibody or antigen binding portion thereof tohuman CD137.

In any of the foregoing aspects, the antibody or antigen binding portionthereof comprises a heavy chain CDR3 comprising the amino acid sequenceDXPFXLDXXYYYYYX (SEQ ID NO: 128), wherein X is any amino acid. In someaspects, mutation of residues D95, L100, Y100E, Y100G, Y100H, orcombinations thereof, of the heavy chain CDR3 of the antibody or antigenbinding portion described herein, results in loss of binding to humanCD137. In some aspects, mutation of residues P97, F98, D100A, Y100D,Y100F, or combinations thereof, of the heavy chain CDR3 of the antibodyor antigen binding portion described herein, to alanine results inreduction of binding to human CD137. In other aspects, mutation ofresidues P97, F98, D100A, Y100D, Y100F, or combinations thereof, of theheavy chain CDR3 of the antibody or antigen binding portion describedherein, to any residue except alanine, results in an increase in bindingto human CD137.

In any of the foregoing aspects, the antibody or antigen binding portionthereof, binds human CD137 with an (K_(D)) of about 45-95 nM, 50-90 nM,55-85 nM, 60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM.In any of the foregoing aspects, the antibody or antigen binding portionthereof, binds human CD137 with an (K_(D)) of about 45 nM to about 95nM, about 50 to about 90 nM, about 55 to about 85 nM, about 60 to about80 nM, about 65 to about 75 nM, about 55 to about 75 nM, about 40 toabout 70 nM, about 50 to about 80 nM, or about 60 to about 90 nM.

In any of the foregoing aspects, the antibody or antigen binding portionthereof comprises heavy and light chain CDRs, wherein heavy chain CDR3comprises the amino acid sequence set forth in SEQ ID NO: 68.

In any of the foregoing aspects, the antibody or antigen binding portionthereof comprises heavy and light chain CDRs selected from the groupconsisting of:

(a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively; and

(b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 108 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively.

In any of the foregoing aspects, the antibody or antigen binding portionthereof comprises heavy and light chain variable regions, wherein theheavy chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 4 and 101; and wherein thelight chain variable region comprises an amino acid sequence of SEQ IDNO: 6.

In any of the foregoing aspects, the antibody or antigen binding portionthereof comprises heavy and light chain variable regions, comprisingamino acid sequences selected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively; and

(b) SEQ ID NO: 101 and 6, respectively.

In any of the foregoing aspects, the antibody or antigen binding portionthereof comprises heavy and light chain variable regions, wherein theheavy chain variable region comprises an amino acid sequence which is atleast 90% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4 and 101; and wherein the light chainvariable region comprises an amino acid sequence which is at least 90%identical to the amino acid sequence of SEQ ID NO: 6.

In any of the foregoing aspects, the antibody or antigen binding portionthereof comprises heavy and light chain variable regions comprisingamino acid sequences at least 90% identical to the amino acid sequencesselected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively; and

(b) SEQ ID NO: 101 and 6, respectively.

In any of the foregoing aspects, the antibody or antigen binding portioncomprises heavy and light chains comprising amino acid sequencesselected from the group consisting of:

(a) SEQ ID NOs: 129 and 133, respectively; and

(b) SEQ ID NOs: 131 and 133, respectively.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof described herein, is an agonist of humanCD137 activity.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof described herein, competes with mAb1 oran antigen binding fragment of mAb1, for binding to the epitope of humanCD137.

In some aspects, the disclosure provides an isolated monoclonal antibodythat specifically binds CD137, or an antigen binding portion thereof,wherein the antibody or antigen binding portion thereof comprises heavyand light chain CDRs selected from the group consisting of:

(a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 70, 79 and 90, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 71, 80 and 91, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 72, 81 and 92, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 73, 82 and 91, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 74, 83 and 93, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 75, 84 and 91, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 74, 85 and 94, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 76, 86 and 95, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 77, 87 and 93, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 88 and 90, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 57 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(m) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 58 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(n) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 59 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(o) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 60 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(p) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 61 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(q) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 58 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(r) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 62 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(s) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:52, 63 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(t) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 64 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(u) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 65 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(v) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 108 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(w) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:107, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively; and

(x) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 109, 110 and 92, respectively.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain variable regions, wherein the heavychain variable region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 101 and 103; and wherein the light chain variable region comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 105.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion comprisesheavy and light chain variable regions encoded by nucleotide sequencesselected from the group consisting of:

(a) SEQ ID NOs: 5 and 7, respectively; and

(b) SEQ ID NOs: 102 and 7, respectively.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain variable regions encoded by nucleotidesequences selected from the group consisting of:

(a) SEQ ID NO: 5 and 7, respectively;

(b) SEQ ID NO: 5 and 29, respectively;

(c) SEQ ID NO: 5 and 31, respectively;

(d) SEQ ID NO: 5 and 33, respectively;

(e) SEQ ID NO: 5 and 35, respectively;

(f) SEQ ID NO: 5 and 37, respectively;

(g) SEQ ID NO: 5 and 39, respectively;

(h) SEQ ID NO: 5 and 41, respectively;

(i) SEQ ID NO: 5 and 43, respectively;

(j) SEQ ID NO: 5 and 45, respectively;

(k) SEQ ID NO: 5 and 47, respectively;

(l) SEQ ID NO: 9 and 7, respectively;

(m) SEQ ID NO: 11 and 7, respectively;

(n) SEQ ID NO: 13 and 7, respectively;

(o) SEQ ID NO: 15 and 7, respectively;

(p) SEQ ID NO: 17 and 7, respectively;

(q) SEQ ID NO: 19 and 7, respectively;

(r) SEQ ID NO: 21 and 7, respectively;

(s) SEQ ID NO: 23 and 7, respectively;

(t) SEQ ID NO: 25 and 7, respectively;

(u) SEQ ID NO: 27 and 7, respectively;

(v) SEQ ID NO: 102 and 7, respectively;

(w) SEQ ID NO: 104 and 7, respectively; and

(x) SEQ ID NO: 5 and 106, respectively.

In yet other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain CDRs, wherein heavy chain CDR3 comprisesthe amino acid sequence set forth in SEQ ID NO: 68.

In another aspect, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain CDRs, wherein heavy chain CDR3 comprisesthe amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X isany amino acid. In some aspects, X is any amino acid except for alanine.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain CDRs, wherein heavy chain CDR3 comprisesthe amino acid sequence DXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X isany amino acid. In some aspects, X is any amino acid except for alanine.

In yet other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain CDRs, wherein heavy chain CDR3 comprisesthe amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X isany amino acid, and wherein mutation of residues D95, L100, Y100E,Y100G, Y100H, or combinations thereof, results in loss of binding tohuman CD137.

In other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain CDRs, wherein heavy chain CDR3 comprisesthe amino acid sequence DXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X isany amino acid, and wherein mutation of residues P97, F98, D100A, Y100D,Y100F, or combinations thereof to alanine results in reduction ofbinding to human CD137.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain CDRs, wherein heavy chain CDR3 comprisesthe amino acid sequence DXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X isany amino acid, and wherein mutation of residues P97, F98, D100A, Y100D,Y100F, or combinations thereof to any residue except alanine, results inan increase in binding to human CD137.

In yet other aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain CDRs, wherein heavy chain CDR3 comprisesthe amino acid sequence DX1X2X3X4LX5X6X7X8YX9YYX10 (SEQ ID NO: 128)wherein X1 is any amino acid, wherein X2 is a non-polar amino acid,wherein X3 is a non-polar amino acid, wherein X4 is any amino acid,wherein X5 is a polar amino acid, wherein X6 is any amino acid, whereinX7 is any amino acid, wherein X8 is a polar amino acid, wherein X9 is apolar amino acid, and wherein X10 is any amino acid. In some aspects,wherein X2 is proline, wherein X3 is phenylalanine or tryptophan,wherein X5 is aspartic acid or glutamic acid wherein X8 is tyrosine, andwherein X9 is tyrosine.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain variable regions comprising amino acidsequences selected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively;

(b) SEQ ID NO: 4 and 28, respectively;

(c) SEQ ID NO: 4 and 30, respectively;

(d) SEQ ID NO: 4 and 32, respectively;

(e) SEQ ID NO: 4 and 34, respectively;

(f) SEQ ID NO: 4 and 36, respectively;

(g) SEQ ID NO: 4 and 38, respectively;

(h) SEQ ID NO: 4 and 40, respectively;

(i) SEQ ID NO: 4 and 42, respectively;

(j) SEQ ID NO: 4 and 44, respectively;

(k) SEQ ID NO: 4 and 46, respectively;

(l) SEQ ID NO: 8 and 6, respectively;

(m) SEQ ID NO: 10 and 6, respectively;

(n) SEQ ID NO: 12 and 6, respectively;

(o) SEQ ID NO: 14 and 6, respectively;

(p) SEQ ID NO: 16 and 6, respectively;

(q) SEQ ID NO: 18 and 6, respectively;

(r) SEQ ID NO: 20 and 6, respectively;

(s) SEQ ID NO: 22 and 6, respectively;

(t) SEQ ID NO: 24 and 6, respectively;

(u) SEQ ID NO: 26 and 6, respectively;

(v) SEQ ID NO: 101 and 6, respectively;

(w) SEQ ID NO: 103 and 6, respectively; and

(x) SEQ ID NO: 4 and 105, respectively.

In other aspects, the disclosure provides, an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137 wherein the antibody or antigen binding portion thereofcomprises heavy and light chain variable regions, wherein the heavychain variable region comprises an amino acid sequence which is at least90% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 101and 103; and wherein the light chain variable region comprises an aminoacid sequence which is at least 90% identical to the amino acid sequenceselected from the group consisting of SEQ ID NOs: 6, 28, 30, 32, 34, 36,38, 40, 42, 44, 46 and 105.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain variable regions comprising amino acidsequences at least 90% identical to the amino acid sequences selectedfrom the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively;

(b) SEQ ID NO: 4 and 28, respectively;

(c) SEQ ID NO: 4 and 30, respectively;

(d) SEQ ID NO: 4 and 32, respectively;

(e) SEQ ID NO: 4 and 34, respectively;

(f) SEQ ID NO: 4 and 36, respectively;

(g) SEQ ID NO: 4 and 38, respectively;

(h) SEQ ID NO: 4 and 40, respectively;

(i) SEQ ID NO: 4 and 42, respectively;

(j) SEQ ID NO: 4 and 44, respectively;

(k) SEQ ID NO: 4 and 46, respectively;

(l) SEQ ID NO: 8 and 6, respectively;

(m) SEQ ID NO: 10 and 6, respectively;

(n) SEQ ID NO: 12 and 6, respectively;

(o) SEQ ID NO: 14 and 6, respectively;

(p) SEQ ID NO: 16 and 6, respectively;

(q) SEQ ID NO: 18 and 6, respectively;

(r) SEQ ID NO: 20 and 6, respectively;

(s) SEQ ID NO: 22 and 6, respectively;

(t) SEQ ID NO: 24 and 6, respectively;

(u) SEQ ID NO: 26 and 6, respectively;

(v) SEQ ID NO: 101 and 6, respectively;

(w) SEQ ID NO: 103 and 6, respectively; and

(x) SEQ ID NO: 4 and 105, respectively.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain sequences comprising amino acidsequences selected from the group consisting of:

(a) SEQ ID NOs: 129 and 133, respectively; and

(b) SEQ ID NOs: 131 and 133, respectively.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain sequences having amino acid sequencesset forth in SEQ ID NOs: 129 and 133, respectively.

In some aspects, the disclosure provides an isolated monoclonalantibody, or antigen binding portion thereof, that specifically bindshuman CD137, wherein the antibody or antigen binding portion thereofcomprises heavy and light chain sequences having amino acid sequencesset forth in SEQ ID NOs: 131 and 133, respectively

In any of the foregoing aspects, the antibody or antigen binding portionspecifically binds to and agonizes human CD137.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, exhibits at least one or more of thefollowing properties selected from the group consisting of:

(a) induces or enhances dimerization of CD137 trimers;

(b) induces or enhances multimerization of CD137 trimers;

(c) induces or enhances T cell activation;

(d) induces or enhances a cytotoxic T cell response;

(e) induces or enhances T cell proliferation;

(f) induces or enhances cytokine production; and

(g) any combination of properties (a)-(f).

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, exhibits at least one or more of thefollowing properties relative to a reference antibody that binds humanCD137, selected from the group consisting of:

(a) does not induce or enhance intrahepatic T cell activation;

(b) does not induce or enhance intrahepatic T cell proliferation;

(c) does not induce or enhance intrasplenic T cell activation;

(d) does not induce or enhance intrasplenic T cell proliferation;

(e) does not induce or enhance macrophage activation;

(f) does not induce or enhance macrophage differentiation;

(g) does not induce or enhance alanine aminotransferase (ALT) activity;and

(h) any combination of properties (a)-(g). In some aspects, thereference antibody is urelumab.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces or enhances humanCD137-mediated T cell activation in the tumor microenvironment, but doesnot significantly induce or enhance human CD137-mediated T cellactivation in the spleen and/or liver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces or enhances T cell activationin the tumor microenvironment, but does not significantly induce orenhance T cell activation in the spleen and/or liver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces or enhances humanCD137-mediated cytotoxic T cell response in the tumor microenvironment,but does not significantly induce or enhance human CD137-mediatedcytotoxic T cell response in the spleen and/or liver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces or enhances a cytotoxic T cellresponse in the tumor microenvironment, but does not significantlyinduce or enhance a T cell response in the spleen and/or liver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces human CD137-mediated T cellproliferation in the tumor microenvironment, but does not significantlyinduce human CD137-mediated T cell proliferation in the spleen and/orliver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces T cell proliferation in thetumor microenvironment, but does not significantly induce T cellproliferation in the spleen and/or liver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces human CD137-mediated T cellinfiltration in the tumor microenvironment, but does not significantlyinduce human CD137-mediated T cell infiltration in the spleen and/orliver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, induces T cell infiltration in thetumor microenvironment, but does not significantly induce T cellinfiltration in the spleen and/or liver.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding fragment thereof, induces or enhances humanCD137-mediated cytokine production in the tumor microenvironment, butdoes not significantly induce or enhance human CD137-mediated cytokineproduction in the spleen and/or liver.

In any of the foregoing aspects, the properties of the antibody orantigen binding portion described herein, are not Fc gamma receptorbinding dependent. In some aspects, the properties of the antibody orantigen binding portion described herein, are enhanced by Fc gammareceptor binding.

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof cross competes with mAb1 (i.e., anantibody comprising the heavy and light chain variable sequences of SEQID NOs: 4 and 6, respectively). In some aspects, the isolated monoclonalantibody or antigen binding portion thereof cross competes with mAb1(i.e., an antibody comprising the heavy and light chain variablesequences of SEQ ID NOs: 4 and 6, respectively), mab8 (i.e., an antibodycomprising the heavy and light chain variable sequences of SEQ ID NOs:101 and 6, respectively) or mAb10 (i.e., an antibody comprising theheavy and light chain variable sequences of SEQ ID NOs: 26 and 6,respectively). In some aspects, the isolated monoclonal antibody orantigen binding portion thereof cross competes with mab8 (i.e., anantibody comprising the heavy and light chain variable sequences of SEQID NOs: 101 and 6, respectively). In some aspects, the isolatedmonoclonal antibody or antigen binding portion thereof cross competeswith mAb10 (i.e., an antibody comprising the heavy and light chainvariable sequences of SEQ ID NOs: 26 and 6, respectively).

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof comprises at least the functionalproperties of mAb1 (i.e., an antibody comprising the heavy and lightchain variable sequences of SEQ ID NOs: 4 and 6, respectively). In someaspects, the isolated monoclonal antibody or antigen binding portionthereof comprises at least the functional properties of mAb1 (i.e., anantibody comprising the heavy and light chain variable sequences of SEQID NOs: 4 and 6, respectively), mab8 (i.e., an antibody comprising theheavy and light chain variable sequences of SEQ ID NOs: 101 and 6,respectively) or mAb10 (i.e., an antibody comprising the heavy and lightchain variable sequences of SEQ ID NOs: 26 and 6, respectively). In someaspects, the isolated monoclonal antibody or antigen binding portionthereof comprises at least the functional properties of mab8 (i.e., anantibody comprising the heavy and light chain variable sequences of SEQID NOs: 101 and 6, respectively). In some aspects, the isolatedmonoclonal antibody or antigen binding portion thereof comprises atleast the functional properties of mAb10 (i.e., an antibody comprisingthe heavy and light chain variable sequences of SEQ ID NOs: 26 and 6,respectively).

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof has a K_(D) value at least equivalent tomAb1 (i.e., an antibody comprising the heavy and light chain variablesequences of SEQ ID NOs: 4 and 6, respectively). In some aspects, theisolated monoclonal antibody or antigen binding portion thereof has aK_(D) value at least equivalent to mAb1 (i.e., an antibody comprisingthe heavy and light chain variable sequences of SEQ ID NOs: 4 and 6,respectively), mab8 (i.e., an antibody comprising the heavy and lightchain variable sequences of SEQ ID NOs: 101 and 6, respectively) ormAb10 (i.e., an antibody comprising the heavy and light chain variablesequences of SEQ ID NOs: 26 and 6, respectively). In some aspects, theisolated monoclonal antibody or antigen binding portion thereof has aK_(D) value at least equivalent to mab8 (i.e., an antibody comprisingthe heavy and light chain variable sequences of SEQ ID NOs: 101 and 6,respectively). In some aspects, the isolated monoclonal antibody orantigen binding portion thereof has a K_(D) value at least equivalent tomAb10 (i.e., an antibody comprising the heavy and light chain variablesequences of SEQ ID NOs: 26 and 6, respectively).

In any of the foregoing aspects, the isolated monoclonal antibody orantigen binding portion thereof, cross-reacts with cynomolgus CD137and/or mouse CD137.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof, is selected from the group consistingof an IgG1, an IgG2, and IgG3, an IgG4, and IgM, and IgA1, and IgA2, andIgD, and an IgE antibody. In some aspects, the isolated monoclonalantibody, or antigen binding portion thereof, is an IgG1 antibody orIgG4 antibody.

In any of the foregoing aspects, the isolated monoclonal antibodycomprises a wild-type IgG1 or wild-type IgG4 heavy chain constantregion. In some aspects, the isolated monoclonal antibody comprises amutant IgG1 heavy chain constant region. In some aspects, the isolatedmonoclonal antibody comprises a mutant IgG4 heavy chain constant region.In some aspects, the mutant IgG4 heavy chain constant region comprises asubstitution at Ser228. In some aspects, the mutant IgG4 heavy chainconstant region comprises substitution S228P.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof, binds to an epitope of CD137, whereinthe amino acid residues comprising the epitope bound by the antibody arelocated within 4 angstroms of the amino acid residues comprising theparatope of the mAb1 antibody, described herein.

In any of the foregoing aspects, the isolated monoclonal antibody, orantigen binding portion thereof, binds to an epitope of CD137, wherein amutation of the epitope bound by the antibody inhibits, reduces, orblocks binding to both the antibody and to antibody mAb1.

In any of the foregoing aspects, the isolated antibody, or antigenbinding portion thereof, is fully human or humanized (i.e., a fullyhuman or humanized antibody or antigen binding portion thereof).

In some aspects, the disclosure provides a pharmaceutical compositioncomprising an isolated monoclonal antibody or antigen binding portionthereof, as described herein, and a pharmaceutically acceptable carrier.

In other aspects, the disclosure provides a nucleic acid comprising anucleotide sequence encoding the light chain, heavy chain, or both lightand heavy chains of an isolated monoclonal antibody, or antigen bindingportion thereof, described herein. In some aspects, the nucleic acidcomprises SEQ ID NOs: 5 and 7. In some aspects, the nucleic acidcomprises SEQ ID NOs: 102 and 7. In some aspects, the disclosureprovides an expression vector comprising the nucleic acid describedherein. In other aspects, the disclosure provides a cell transformedwith an expression vector described herein.

In another aspect, the disclosure provides a method for producing anisolated monoclonal antibody, or antigen binding portion thereof, thatspecifically binds human CD137, the method comprising maintaining a celldescribed herein under conditions permitting expression of themonoclonal antibody or antigen binding portion thereof. In some aspects,the method for producing the monoclonal antibody that specifically bindshuman CD137, or antigen binding portion thereof, further comprisesobtaining the monoclonal antibody or antigen binding portion thereof.

In yet another aspect, the disclosure provides a method for inducing orenhancing dimerization of human CD137 trimers in a subject, comprisingadministering to a subject in need thereof, an effective amount of anisolated monoclonal antibody, or antigen binding portion thereof, asdescribed herein, or a pharmaceutical composition described herein.

In another aspect, the disclosure provides a method for inducing orenhancing multimerization of human CD137 trimers in a subject,comprising administering to a subject in need thereof, an effectiveamount of an isolated monoclonal antibody, or antigen binding portionthereof, as described herein, or a pharmaceutical composition describedherein.

In other aspects, the disclosure provides a method for inducing orenhancing T cell activation mediated by human CD137 in a subject,comprising administering to a subject in need thereof, an effectiveamount of an isolated monoclonal antibody, or antigen binding portionthereof, as described herein, or a pharmaceutical composition describedherein. In some aspects, T cell activation occurs in a tumormicroenvironment. In other aspects, T cell activation does notsignificantly occur in the spleen and/or liver of the subject.

In another aspect, the disclosure provides a method for inducing orenhancing a cytotoxic T cell response mediated by human CD137 in asubject, comprising administering to a subject in need thereof, aneffective amount of an isolated monoclonal antibody, or antigen bindingportion thereof, as described herein, or a pharmaceutical compositiondescribed herein. In some aspects, the cytotoxic T cell response occursin a tumor microenvironment. In other aspects, the cytotoxic T cellresponse does not significantly occur in the spleen and/or liver of thesubject.

In some aspects, the disclosure provides a method for inducing orenhancing cytokine production mediated by human CD137 in a subject,comprising administering to a subject in need thereof, an effectiveamount of an isolated monoclonal antibody, or antigen binding portionthereof, as described herein, or a pharmaceutical composition describedherein. In some aspects, the cytokine produced is IL-2, TNFα, IL-13,IFNγ, or combinations thereof. In some aspects, the cytokine produced isIL-2. In some aspects, the cytokine produced is TNFα. In some aspects,the cytokine produced is IL-13. In some aspects, the cytokine producedis IFNγ. In some aspects, the cytokine produced is IL-2 and TNFα. Insome aspects, the cytokine produced is IL-2 and IL-13. In some aspects,the cytokine produced is IL-2 and IFNγ. In some aspects, the cytokineproduced is TNFα and IL-13. In some aspects, the cytokine produced isTNFα and IFNγ. In some aspects, the cytokine produced is IL-13 and IFNγ.In some aspects, the cytokine produced is IL-2, TNFα and IL-13. In someaspects, the cytokine produced is IL-2, TNFα and IFNγ. In some aspects,the cytokine produced is IFNγ TNFα and IL-13. In other aspects, cytokineproduction occurs in a tumor microenvironment. In yet other aspects,cytokine production does not significantly occur in the spleen and/orliver of the subject.

In another aspect, the disclosure provides a method for inducing orenhancing T cell proliferation mediated by human CD137 in a subject,comprising administering to a subject in need thereof, an effectiveamount of an isolated monoclonal antibody, or antigen binding portionthereof, as described herein, or a pharmaceutical composition describedherein. In some aspects, T cell proliferation occurs in a tumormicroenvironment. In other aspects, T cell proliferation does notsignificantly occur in the spleen and/or liver of the subject.

In another aspect, the disclosure provides a method for reducing orinhibiting tumor growth, comprising administering to a subject in needthereof, an effective amount of an isolated monoclonal antibody, orantigen binding portion thereof, as described herein, or apharmaceutical composition described herein.

In yet another aspect, the disclosure provides a method for treating adisorder mediated by human CD137 in a subject, comprising administeringto a subject in need thereof, an effective amount of an isolatedmonoclonal antibody, or antigen binding portion thereof, as describedherein, or a pharmaceutical composition described herein.

In some aspects, the disclosure provides a method for treating cancer ina subject, comprising administering to a subject in need thereof, aneffective amount of an isolated monoclonal antibody, or antigen bindingportion thereof, as described herein, or a pharmaceutical compositiondescribed herein. In some aspects, the cancer is selected from the groupconsisting of melanoma, glioma, renal, breast, hematological and headand neck cancer. In some aspects, the hematological cancer is a B celllymphoma.

In some aspects, the disclosure provides a method of inducing ananti-tumor memory immune response, comprising administering to a subjectin need thereof, an effective amount of an isolated monoclonal antibody,or antigen binding portion thereof, as described herein, or apharmaceutical composition described herein.

In any of the foregoing aspects, infiltration of immune cells into atumor microenvironment is increased after administration of an antibodyor antigen binding portion. In some aspects, immune cells express CD45.

In any of the foregoing aspects, quantity of T regulatory (Treg) cellsis reduced in a tumor microenvironment after administration of anantibody or antigen binding portion. In some aspects, Treg cells expressCD4, FOXP-3 and CD24.

In any of the foregoing aspects, quantity of macrophages cells isreduced in a tumor microenvironment after administration of a monoclonalantibody or antigen binding portion. In some aspects, macrophagesexpress CD45 and CD1 lb.

In any of the foregoing aspects, T cell exhaustion is reduced afteradministration of an antibody or antigen binding portion. In someaspects, reduction of T cell exhaustion comprises a decrease inexpression of TIGIT, PD-1, LAG-3 or a combination thereof. In someaspects, reduction of T cell exhaustion comprises a decrease inexpression of TIGIT and PD-1.

In any of the foregoing aspects, depletion of CD4+ T cells, CD8+ Tcells, Natural Killer cells, or combinations thereof, reduces theefficacy of the antibody or antigen binding portion thereof.

In another aspect, the disclosure provides a method for detecting thepresence or absence of human CD137 in a biological sample, comprising:

(a) contacting a biological sample with an antibody or antigen-bindingportion described herein, wherein the antibody or antigen-bindingportion is labeled with a detectable substance; and

(b) detecting the antibody or antigen-binding portion bound to humanCD137 to thereby detect the presence or absence of human CD137 in thebiological sample.

In another aspect, the disclosure provides a kit comprising a containercomprising an antibody or antigen-binding portion described herein, andan optional pharmaceutically acceptable carrier, or a pharmaceuticalcomposition described herein, and a package insert comprisinginstructions for administration of the antibody or pharmaceuticalcomposition, for treating or delaying progression of cancer or reducingor inhibiting tumor growth in a subject in need thereof.

In another aspect, the disclosure provides a kit comprising a containercomprising an antibody or antigen-binding portion described herein, andan optional pharmaceutically acceptable carrier, or a pharmaceuticalcomposition described herein, and a package insert comprisinginstructions for administration of the antibody or pharmaceuticalcomposition alone or in combination with another agent, for treating ordelaying progression of cancer or reducing or inhibiting tumor growth ina subject in need thereof.

In another aspect, the disclosure provides use of an isolated monoclonalantibody, or antigen binding portion thereof, as described herein, toinduce or enhance T cell activation mediated by human CD137 in asubject. In other aspects, the disclosure provides use of an isolatedmonoclonal antibody, or antigen binding portion thereof, as describedherein, to induce or enhance multimerization of human CD137 trimers in asubject. In another aspect, the disclosure provides use of an isolatedmonoclonal antibody, or antigen binding portion thereof, as describedherein, to induce or enhance a cytotoxic T cell response mediated byhuman CD137 in a subject. In other aspects, the disclosure provides useof an isolated monoclonal antibody, or antigen binding portion thereof,as described herein, to induce or enhance cytokine production mediatedby human CD137 in a subject. In another aspect, the disclosure providesuse of an isolated monoclonal antibody, or antigen binding portionthereof, as described herein, to induce or enhance T cell proliferationmediated by human CD137 in a subject.

In another aspect, the disclosure provides use of an isolated monoclonalantibody, or antigen binding portion thereof, as described herein, toreduce or inhibit tumor growth in a subject in need thereof. In otheraspects, the disclosure provides use of an isolated monoclonal antibody,or antigen binding portion thereof, as described herein, to treat adisorder mediated by human CD137 in a subject in need thereof. Inanother aspect, the disclosure provides use of an isolated monoclonalantibody, or antigen binding portion thereof, as described herein, totreat cancer in a subject in need thereof.

In another aspect, the disclosure provides use of an isolated monoclonalantibody, or antigen binding portion thereof, as described herein, forthe manufacture of a medicament for treating or delaying progression ofcancer or reducing or inhibiting tumor growth in a subject in needthereof. In other aspects, the disclosure provides an isolatedmonoclonal antibody, or antigen binding portion thereof, as describedherein, in the manufacture of a medicament for treating or delayingprogression of cancer or reducing or inhibiting tumor growth in asubject in need thereof. In another aspect, the disclosure provides anisolated monoclonal antibody or antigen binding portion thereof, asdescribed herein, for use as a medicament.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 provides graphs depicting the distribution of binding affinitiesof affinity matured clones of the parental anti-CD137 antibody mAb1.

FIG. 2 provides a schematic showing the results of mAb1 CDRH3 alaninescanning, as measured by binding affinity (K_(D)) to human or mouseCD137.

FIG. 3A shows the amino acid sequence of human CD137 (residues 24-159 ofSEQ ID NO: 3) wherein residues comprising an epitope bound by mAb1, mAb4or mAb5 are indicated in bold.

FIG. 3B is a graph depicting kinetic binding data of mAb1 to theextracellular domain of mouse and rat CD137 as determined by surfaceplasmon resonance.

FIG. 3C provides x-ray crystallography images of human CD137 bound toCD137L (shown in grey) and residues E111, T113, K114 and P135 shown asspheres.

FIG. 3D provides x-ray crystallography images of human CD137 bound toCD137L (shown in grey) in trimeric formation, and residues E111, T113,K114 and P135 shown as spheres.

FIG. 4A provides a scatterplot of flow cytometric data depicting anincrease in TIGIT (top) or PD-1 (bottom) expression on CD44+ T cells inresponse to anti-CD137 antibodies.

FIG. 4B provides graphs depicting the quantification of CD8+CD44+ Tcells expressing TIGIT (top) or PD-1 (bottom) in the spleen of miceafter treatment with anti-CD137 antibodies.

FIG. 4C provides graphs depicting the quantification of CD8+ T cells inthe spleen of mice after treatment with anti-CD137 antibodies, aspercentage of CD45+ cells (left) or cell number per spleen (right).

FIG. 5A provides graphs showing individual CT26 tumor volumes in miceafter treatment with anti-CD137 antibodies at indicated dosages.

FIG. 5B is a graph showing the mean tumor volumes provided in FIG. 5A.

FIG. 5C is a Kaplan-Meier graph showing overall survival of mice withtumors after treatment with anti-CD137 antibodies.

FIG. 5D is a graph showing tumor volume in mice re-challenged withtumorigenic CT26 cells.

FIG. 6A provides graphs showing individual CT26 tumor volumes in miceafter treatment with parental and affinity-matured anti-CD137antibodies.

FIG. 6B is a graph providing the mean tumor volumes provided in FIG. 6A.

FIG. 7 provides graphs depicting the percentage of CD8+ or CD4+ T cells,from splenic T cells (top) and tumor infiltrating leukocytes (bottom)after treatment with anti-CD137 antibodies at indicated dosages.

FIG. 8 provides graphs showing individual tumor volumes when mice weretreated with mAb1, with or without lymphocyte depleting antibodies. CD4+T cells were depleted with GK1.5 (middle graph), CD8+ T cells weredepleted with YTS169.4 (second graph from the right), and NK cells weredepleted with an anti-asialo-GM1 antibody (last graph on the right).

FIG. 9 provides graphs showing individual tumor volumes in mice havingeither CT26 tumors (colon carcinoma), EMT-6 tumors (breast carcinoma),A20 tumors (B cell lymphoma), or MC38 tumors (colon carcinoma) andtreated with mAb8 or isotype control antibody.

FIGS. 10A-10C show the in vivo anti-tumor efficacy of anti-CD137antibodies administered at 150 μg/mouse. Individual tumor volumes areshown in 10A, mean tumor volumes are shown in 10B and percent survivalis shown in 10C.

FIGS. 11A-11C show the in vivo anti-tumor efficacy of anti-CD137antibodies administered at 20 μg/mouse. Individual tumor volumes areshown in 11A, mean tumor volumes are shown in 11B and percent survivalis shown in 11C.

FIG. 12 provides graphs showing individual tumor volumes in mice havingCT26 tumors and treated with varying doses of mAb1 (i.e., 12.5, 25, 50,100 or 200 μg) or isotype control.

FIGS. 13A and 13B show the contribution of Fc binding in the anti-tumorefficacy of mAb1. FIG. 13A shows mAb1 as an IgG4 isotype or an IgG4aglycosylated isotype. Mean tumor volumes are shown on the top andindividual tumor volumes are shown on the bottom. FIG. 13B shows mAb1 asan IgG4 isotype or an IgG1 aglycosylated isotype. Mean tumor volumes areshown on the top and individual tumor volumes are shown on the bottom.

FIGS. 14A-14D show the in vivo anti-tumor efficacy of anti-CD137antibodies in mice with large established tumors (i.e., 500 mm³) priorto receiving treatment. Individual tumor volumes are shown in 14A and14D, mean tumor volumes are shown in 14B and percent survival is shownin 14C.

FIG. 15 provides a Kaplan-Meier survival graph showing protectiveanti-tumor immunity in mice previously treated with mAb1, mAb8 orisotype control from FIGS. 14A-14C and considered cured, re-challengedwith CT26 cells in an opposing flank.

FIG. 16A provides scatterplots of flow cytometric data showing theexpansion of CD45+ intrahepatic T cells following treatment withanti-CD137 antibodies at indicated dosages.

FIG. 16B provides graphs depicting the quantification of intrahepaticCD8+ T cells (left) and CD4+ T cells (right) following treatment withanti-CD137 antibodies at indicated dosages.

FIG. 17A provides graphs depicting the percentage of CD3+, CD4+, or CD8+T cells, from splenic T cells after treatment of mice withaffinity-matured anti-CD137 antibodies.

FIG. 17B provides graphs depicting the percentage of CD3+, CD4+, or CD8+T cells from liver T cells after treatment of mice with affinity-maturedanti-CD137 antibodies.

FIG. 18A provides graphs depicting the percentage of splenic CD8+CD44+ Tcells expressing TIGIT, PD-1, or LAG3 after treatment of mice withaffinity-matured anti-CD137 antibodies.

FIG. 18B provides graphs depicting the percentage of liver CD8+CD44+ Tcells expressing TIGIT, PD-1, or LAG3 after treatment of mice withaffinity-matured anti-CD137 antibodies.

FIG. 19A provides graphs depicting the percentage of splenic CD4+CD44+ Tcells expressing TIGIT, PD-1, or LAG3 after treatment of mice withaffinity-matured anti-CD137 antibodies.

FIG. 19B provides graphs depicting the percentage of liver CD4+CD44+ Tcells expressing TIGIT, PD-1, or LAG3 after treatment of mice withaffinity-matured anti-CD137 antibodies.

FIGS. 20A-20C provide graphs of in vivo indicators of toxicity resultingfrom multiple administrations of anti-CD137 antibodies mAb1, mAb8 or 3H3at varying doses. FIG. 20A is a graph showing percentage of CD8+ T cellsin the liver after administration of the anti-CD137 antibodies. FIG. 20Bis a graph showing alanine aminotransferase (ALT) activity in the plasmaof mice administered anti-CD137 antibodies. FIG. 20C is a graph showingthe levels of TNFα in the plasma of mice administered anti-CD137antibodies.

FIG. 21 provides representative images of sectioned livers stained withhematoxylin and eosin (H&E) from mice treated with mAb1, mAb8, 3H3 orisotype control as described in FIGS. 20A-20C. Arrows indicateinfiltration of immune cells.

FIGS. 22A-22D provide representative FACS plots showing immune cellreprogramming in the tumor microenvironment. Mice having CT26 tumorswere administered multiple doses of mAb8 or isotype control (days 0, 3,6 and 9). FIG. 22A shows overall immune cell infiltration based on CD45expression. FIG. 22B shows reduction in Treg cells as measured by FOXP-3and CD25 expression. FIG. 22C shows reduction of T-cell exhaustion asmeasured by PD-1 and TIGIT expression. FIG. 22D shows reduction oftumor-associated macrophages as measured by F4/80 and CD11b expression.

FIG. 23 shows immunophenotyping analysis of spleens from mice havingCT26 tumors and treated with either anti-CD137 antibodies mAb1 and 3H3,or isotype control.

FIG. 24 is a graph showing the concentration of IL-2 (pg/ml) produced bymurine T cells in an OVA stimulation assay, when stimulated with theanti-CD137 antibodies indicated. Along with Atezolizumab (anti-PD-L1antibody), a murine anti-PD-1 (RMP1-14) was used as a comparator.

FIGS. 25A and 25B are graphs showing the percentage of murine CD8+ Tcells expressing either CD25 (25A) or TIGIT (25B) when stimulated withthe anti-CD137 antibodies indicated, in an OVA stimulation assay. Alongwith Atezolizumab (anti-PD-L1 antibody), a murine anti-PD-1 (RMP1-14)and murine anti-CD137 (3H3) were used as comparators.

FIG. 26 provides bar graphs depicting the quantification of cytokines(IL-2, TNFα, IL-13, and IFNγ) produced by CD3+ T cells followingincubation with plate-bound anti-CD137 antibodies. Cytokine levels areshown as fold increase over baseline activation by an anti-CD3 antibody.

FIGS. 27A-27C provide graphs depicting the dose-response of IFNγproduction in a mixed lymphocyte reaction following treatment withanti-CD137 antibodies. An anti-PD1 antibody (Keytruda; Merck) was usedas a control.

FIG. 28 is a graph showing IFNγ production from human T cellsco-cultured with CHO cells engineered to express CD32 (CHO-CD32 cells)in the presence of anti-CD137 antibodies mAb1, mAb8, mAb4 or mAb5, orisotype control.

FIG. 29 is a graph showing proliferation of Treg cells when co-culturedwith CHO cells engineered to express CD32 (CHO-CD32 cells) in thepresence or absence of anti-CD137 antibodies mAb1, mAb8, mAb4 or mAb5,isotype control.

FIG. 30 provides graphs showing NFκβ and SRF signaling in CCL-119 cellstransduced with luciferase reporters for NFκβ or SRF in the presence ofmAb1, mAB8, mAb4 or mAb5 at varying concentrations.

FIG. 31 provides graphs showing induction of IL-6, TNFα, or IL-27 bybone marrow-derived mouse macrophages stimulated with TLR9 agonist CpGin the presence of anti-CD137 antibodies mAb1, 3H3 or LOB12.3, orisotype control.

FIG. 32 provides a graph showing induction of TNFα by human monocytederived macrophages stimulated with LPS in the presence of anti-CD137antibodies mAb1, mAb4 or mAb5, or isotype control.

FIG. 33 provides a graph showing effect of anti-CD137 antibodies onmacrophage differentiation as determined by CD64 expression of THP1monocytes cultured with PMA in the presence of anti-CD137 antibodiesmAb1, mAb4 or mAb5, or isotype control.

FIGS. 34A-34C provides graphs showing percentage of hCD45+, hCD8+ orhCD4+ from immunocompetent mice that received human PBMCs and anti-CD137antibodies mAb1, mAb4 or mAb5, or isotype control.

DETAILED DESCRIPTION

Cancer therapy with agonist anti-CD137 antibodies has been shown toinduce immune-mediated tumor rejections in mice, and analogous agents ofthis kind are currently being tested in cancer patients. Previousreports have indicated that administration of anti-CD137 antibodies caninduce significant accumulations of polyclonal infiltrates of Tlymphocytes in the liver (Dubrot et al., (2010) Cancer Immunology,Immunotherapy 59(8):1223-1233), suggestive of hepatic inflammation andthe potential for drug-induced liver toxicity. A recent report on theclinical evaluation of an agonistic anti-CD137 antibody (Urelumab,BMS-663513; Bristol-Myers Squibb) documented the observation oftreatment-related adverse events in human subjects, includingindications of severe hepatotoxicity (transaminitis) correlating withantibody dose (Segal et al., (2016) Clin Cancer Res 23(8):1929-1936).

The present disclosure provides isolated monoclonal antibodies, orantigen binding portions thereof, that specifically bind to an epitopeof human CD137 and agonize human CD137. In some embodiments, theantibody or antigen binding portion thereof competes with mAb1 forbinding to the epitope of human CD137. In some aspects, the anti-CD137agonist antibodies of the disclosure induce cytokine production andexpansion of CD8+ T cells in the tumor microenvironment, and protectiveanti-tumor immunity in vivo with a concomitant reduction in thepotential for toxicity-related events, as compared to the anti-mouseCD137 3H3 antibody (Melero et al. (1997) Nature Medicine 3(6):682-685;Uno et al. (2006) Nature Medicine 12(6):693-696) and to at least twoanti-human CD137 antibodies in clinical development(BMS-663513/Urelumab, Bristol-Meyers Squibb, and PF-05082566/Utomilumab,Pfizer).

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Further, unless otherwiserequired by context, singular terms shall include pluralities and pluralterms shall include the singular.

As used herein, “about” will be understood by persons of ordinary skilland will vary to some extent depending on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill given the context in which it is used, “about” will meanup to plus or minus 10% of the particular value.

As used herein, the term “agonist” refers to any molecule that partiallyor fully promotes, induces, increases, and/or activates a biologicalactivity of a native polypeptide disclosed herein (e.g., CD137).Suitable agonist molecules specifically include agonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. In some embodiments, activation in the presence of theagonist is observed in a dose-dependent manner. In some embodiments, themeasured signal (e.g., biological activity) is at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 100% higher than the signal measured with anegative control under comparable conditions. Also disclosed herein, aremethods of identifying agonists suitable for use in the methods of thedisclosure. For example, these methods include, but are not limited to,binding assays such as enzyme-linked immuno-absorbent assay (ELISA),Forte Bio© systems, and radioimmunoassay (RIA). These assays determinethe ability of an agonist to bind the polypeptide of interest (e.g., areceptor or ligand, e.g., CD137) and therefore indicate the ability ofthe agonist to promote, increase or activate the activity of thepolypeptide. Efficacy of an agonist can also be determined usingfunctional assays, such as the ability of an agonist to activate orpromote the function of the polypeptide. For example, a functional assaymay comprise contacting a polypeptide with a candidate agonist moleculeand measuring a detectable change in one or more biological activitiesnormally associated with the polypeptide. The potency of an agonist isusually defined by its EC₅₀ value (concentration required to activate50% of the agonist response). The lower the EC₅₀ value the greater thepotency of the agonist and the lower the concentration that is requiredto activate the maximum biological response.

As used herein, the term “alanine scanning” refers to a technique usedto determine the contribution of a specific wild-type residue to thestability or function(s) (e.g., binding affinity) of a given protein orpolypeptide. The technique involves the substitution of an alanineresidue for a wild-type residue in a polypeptide, followed by anassessment of the stability or function(s) (e.g., binding affinity) ofthe alanine-substituted derivative or mutant polypeptide and comparisonto the wild-type polypeptide. Techniques to substitute alanine for awild-type residue in a polypeptide are known in the art.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., cancer, includingprophylaxis, lessening in the severity or progression, remission, orcure thereof.

As used herein, the term “amino acid” refers to naturally occurring andsynthetic amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., a carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes. Asused here, a “polar amino acid” refers to an amino acid comprising aside chain that prefers to reside in an aqueous environment. In someembodiments, a polar amino acid is selected from the group consistingof: arginine, asparagine, aspartic acid, glutamic acid, glutamine,histidine, lysine, serine, theronine and tyrosine. Polar amino acids canbe positive, negatively or neutrally charged. As used herein, a“non-polar amino acid” refers to an amino acid selected from the groupconsisting of: alanine, cysteine, glycine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine.

As used herein, an “amino acid substitution” refers to the replacementof at least one existing amino acid residue in a predetermined aminoacid sequence (an amino acid sequence of a starting polypeptide) with asecond, different “replacement” amino acid residue. An “amino acidinsertion” refers to the incorporation of at least one additional aminoacid into a predetermined amino acid sequence. While the insertion willusually consist of the insertion of one or two amino acid residues,larger “peptide insertions,” can also be made, e.g. insertion of aboutthree to about five or even up to about ten, fifteen, or twenty aminoacid residues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

As used herein, the term “amount” or “level” refers to a detectablequantity, level or abundance of a substance (e.g., a protein). Whenreferring to a polypeptide, such as those described herein, the terms“level of expression” or “expression level” in general are usedinterchangeably and generally refer to a detectable amount of apolypeptide in a biological sample (e.g., on the surface of a cell).

As used herein, the term “anti-CD137 agonist antibody” (usedinterchangeably with the term “anti-CD137 antibody”) refers to anantibody that specifically binds to CD137 and partially or fullypromotes, induces, increases, and/or activates CD137 biologicalactivity, response, and/or downstream pathway(s) mediated by CD137signaling or other CD137-mediated function. In some embodiments, ananti-CD137 agonist antibody binds to CD137 and allows binding of CD137L.In some embodiments, an anti-CD137 agonist antibody binds to CD137 andinduces multimerization of CD137. In some embodiments, an anti-CD137agonist antibody binds to CD137 and induces the dimerization of CD137trimers. In some embodiments, an anti-CD137 agonist antibody binds toCD137 and induces the multimerization of CD137 trimers. Examples ofanti-CD137 agonist antibodies are provided herein. Methods for detectingformation of a trimer:trimer complex are known to those of skill in theart. For example, electron microscopy has been shown to detect suchcomplexes, see, e.g., Won, E. The Journal of Biological Chemistry, Vol.285 (12): 9202-9210 (2010)

As used herein, the term “anti-CD137 mAb1” (used interchangeably with“mAb1”) refers to an exemplary anti-CD137 agonist antibody thatcomprises the variable heavy chain (V_(H)) amino acid sequence:

(SEQ ID NO: 4) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS,and the variable light chain (V_(L)) amino acid sequence:

(SEQ ID NO: 6) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHLFPITFGG GTKVEIK.

As used herein, the term “anti-CD137 mAb8” (used interchangeably with“mAb8”) refers to an exemplary anti-CD137 agonist antibody thatcomprises the variable heavy chain ((V_(H)) amino acid sequence:

(SEQ ID NO: 101) EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEWVSAISGSGDTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS;and the variable light chain (V_(L)) amino acid sequence:

(SEQ ID NO: 6) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHLFPITFGG GTKVEIK.

As used herein, the term “anti-CD137 mAb10” (used interchangeably with“mAb10”) refers to an exemplary anti-CD137 agonist antibody thatcomprises the variable heavy chain ((V_(H)) amino acid sequence:

(SEQ ID NO: 26) EVQLLESGGGLVQPGGSLRLSCAASGFTFYGYAMSWVRQAPGKGLEWVAAISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS;and the variable light chain (V_(L)) amino acid sequence:

(SEQ ID NO: 6) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHLFPITFGG GTKVEIK.

As used herein, the term “antibody” refers to a whole antibodycomprising two light chain polypeptides and two heavy chainpolypeptides. Whole antibodies include different antibody isotypesincluding IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody”includes a polyclonal antibody, a monoclonal antibody, a chimerized orchimeric antibody, a humanized antibody, a primatized antibody, adeimmunized antibody, and a fully human antibody. The antibody can bemade in or derived from any of a variety of species, e.g., mammals suchas humans, non-human primates (e.g., orangutan, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be apurified or a recombinant antibody.

As used herein, the terms “antibody fragment,” “antigen-bindingfragment,” “antigen binding portion” or similar terms refer to afragment of an antibody that retains the ability to bind to a targetantigen (e.g., CD137) and inhibit the activity of the target antigen.Such fragments include, e.g., a single chain antibody, a single chain Fvfragment (scFv), an Fd fragment, a Fab fragment, a Fab′ fragment, or anF(ab′)₂ fragment. An scFv fragment is a single polypeptide chain thatincludes both the heavy and light chain variable regions of the antibodyfrom which the scFv is derived. In addition, intrabodies, minibodies,triabodies, and diabodies are also included in the definition ofantibody and are compatible for use in the methods described herein.See, e.g., Todorovska et al., (2001) J. Immunol. Methods 248(1):47-66;Hudson and Kortt, (1999) J. Immunol. Methods 231(1): 177-189; Poljak,(1994) Structure 2(12): 1121-1123; Rondon and Marasco, (1997) Annu. Rev.Microbiol. 51:257-283, the disclosures of each of which are incorporatedherein by reference in their entirety.

As used herein, the term “antibody fragment” also includes, e.g., singledomain antibodies such as camelized single domain antibodies. See, e.g.,Muyldermans et al., (2001) Trends Biochem. Sci. 26:230-235; Nuttall etal., (2000) Curr. Pharm. Biotech. 1:253-263; Reichmann et al., (1999) J.Immunol. Meth. 231:25-38; PCT application publication nos. WO 94/04678and WO 94/25591; and U.S. Pat. No. 6,005,079, all of which areincorporated herein by reference in their entireties. In someembodiments, the disclosure provides single domain antibodies comprisingtwo VH domains with modifications such that single domain antibodies areformed.

In some embodiment, an antigen-binding fragment includes the variableregion of a heavy chain polypeptide and the variable region of a lightchain polypeptide. In some embodiments, an antigen-binding fragmentdescribed herein comprises the CDRs of the light chain and heavy chainpolypeptide of an antibody.

The term “antigen presenting cell” or “APC” is a cell that displaysforeign antigen complexed with MHC on its surface. T cells recognizethis complex using T cell receptor (TCR). Examples of APCs include, butare not limited to, dendritic cells (DCs), peripheral blood mononuclearcells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such asC1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). SomeAPCs internalize antigens either by phagocytosis or by receptor-mediatedendocytosis.

The term “antigen presentation” refers to the process by which APCscapture antigens and enables their recognition by T cells, e.g., as acomponent of an MHC-I and/or MHC-II conjugate.

As used herein, the term “apoptosis” refers to the process of programmedcell death that occurs in multicellular organisms (e.g. humans). Thehighly-regulated biochemical and molecular events that result inapoptosis can lead to observable and characteristic morphologicalchanges to a cell, including membrane blebbing, cell volume shrinkage,chromosomal DNA condensation and fragmentation, and mRNA decay. A commonmethod to identify cells, including T cells, undergoing apoptosis is toexpose cells to a fluorophore-conjugated protein (Annexin V). Annexin Vis commonly used to detect apoptotic cells by its ability to bind tophosphatidylserine on the outer leaflet of the plasma membrane, which isan early indicator that the cell is undergoing the process of apoptosis.

As used herein, the term “binds to immobilized CD137,” refers to theability of a human antibody of the disclosure to bind to CD137, forexample, expressed on the surface of a cell or which is attached to asolid support.

As used herein, the term “bispecific” or “bifunctional antibody” refersto an artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, (1990)Clin. Exp. Immunol. 79:315-321; Kostelny et al., (1992) J. Immunol.148:1547-1553.

Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chain/light-chain pairs have differentspecificities (Milstein and Cuello, (1983) Nature 305:537-539). Antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion of the heavy chain variable regionis preferably with an immunoglobulin heavy-chain constant domain,including at least part of the hinge, CH2, and CH3 regions. For furtherdetails of illustrative currently known methods for generatingbispecific antibodies see, e.g., Suresh et al., (1986) Methods Enzymol.121:210; PCT Publication No. WO 96/27011; Brennan et al., (1985) Science229:81; Shalaby et al., J. Exp. Med. (1992) 175:217-225; Kostelny etal., (1992) J. Immunol. 148(5):1547-1553; Hollinger et al., (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Gruber et al., (1994) J. Immunol.152:5368; and Tutt et al., (1991) J. Immunol. 147:60. Bispecificantibodies also include cross-linked or heteroconjugate antibodies.Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. See, e.g., Kostelny et al. (1992) J Immunol148(5):1547-1553. The leucine zipper peptides from the Fos and Junproteins may be linked to the Fab′ portions of two different antibodiesby gene fusion. The antibody homodimers may be reduced at the hingeregion to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See, e.g., Gruber et al. (1994) J Immunol 152:5368.Alternatively, the antibodies can be “linear antibodies” as describedin, e.g., Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly,these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1)which form a pair of antigen binding regions. Linear antibodies can bebispecific or monospecific.

Antibodies with more than two valencies (e.g., trispecific antibodies)are contemplated and described in, e.g., Tutt et al. (1991) J Immunol147:60.

The disclosure also embraces variant forms of multi-specific antibodiessuch as the dual variable domain immunoglobulin (DVD-Ig) moleculesdescribed in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. TheDVD-Ig molecules are designed such that two different light chainvariable domains (VL) from two different parent antibodies are linked intandem directly or via a short linker by recombinant DNA techniques,followed by the light chain constant domain. Similarly, the heavy chaincomprises two different heavy chain variable domains (VH) linked intandem, followed by the constant domain CH1 and Fc region. Methods formaking DVD-Ig molecules from two parent antibodies are further describedin, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715. In someembodiments, the bispecific antibody is a Fabs-in-Tandem immunoglobulin,in which the light chain variable region with a second specificity isfused to the heavy chain variable region of a whole antibody. Suchantibodies are described in, e.g., International Patent ApplicationPublication No. WO 2015/103072.

As used herein, “cancer antigen” refers to (i) tumor-specific antigens,(ii) tumor-associated antigens, (iii) cells that express tumor-specificantigens, (iv) cells that express tumor-associated antigens, (v)embryonic antigens on tumors, (vi) autologous tumor cells, (vii)tumor-specific membrane antigens, (viii) tumor-associated membraneantigens, (ix) growth factor receptors, (x) growth factor ligands, and(xi) any other type of antigen or antigen-presenting cell or materialthat is associated with a cancer.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. The anti-CD137 antibodiesdescribed herein can be used to treat patients who have, who aresuspected of having, or who may be at high risk for developing any typeof cancer, including renal carcinoma or melanoma. Exemplary carcinomasinclude those forming from tissue of the cervix, lung, prostate, breast,head and neck, colon and ovary. The term also includes carcinosarcomas,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

As used herein the term “compete”, when used in the context ofantigen-binding proteins (e.g., immunoglobulins, antibodies, orantigen-binding fragments thereof) that compete for binding to the sameepitope, refers to a interaction between antigen-binding proteins asdetermined by an assay (e.g., a competitive binding assay; across-blocking assay), wherein a test antigen-binding protein (e.g., atest antibody) inhibits (e.g., reduces or blocks) specific binding of areference antigen-binding protein (e.g., a reference antibody, such asmAb1) to a common antigen (e.g., CD137 or a fragment thereof). In someembodiments, the antibodies described herein cross compete with mAb1(i.e., an antibody comprising the heavy and light chain variablesequences of SEQ ID NOs: 4 and 6, respectively), mab8 (i.e., an antibodycomprising the heavy and light chain variable sequences of SEQ ID NOs:101 and 6, respectively) or mAb10 (i.e., an antibody comprising theheavy and light chain variable sequences of SEQ ID NOs: 26 and 6,respectively).

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence. Polypeptides derived from another peptide may have one ormore mutations relative to the starting polypeptide, e.g., one or moreamino acid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions.

A polypeptide can comprise an amino acid sequence which is not naturallyoccurring. Such variants necessarily have less than 100% sequenceidentity or similarity with the starting molecule. In certainembodiments, the variant will have an amino acid sequence from about 75%to less than 100% amino acid sequence identity or similarity with theamino acid sequence of the starting polypeptide, more preferably fromabout 80% to less than 100%, more preferably from about 85% to less than100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% toless than 100%, e.g., over the length of the variant molecule.

In certain embodiments, there is one amino acid difference between astarting polypeptide sequence and the sequence derived there from.Identity or similarity with respect to this sequence is defined hereinas the percentage of amino acid residues in the candidate sequence thatare identical (i.e., same residue) with the starting amino acidresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. In certainembodiments, a polypeptide consists of, consists essentially of, orcomprises an amino acid sequence selected from a sequence set forth inTable 3 or Table 4. In certain embodiments, a polypeptide includes anamino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto an amino acid sequence selected from a sequence set forth in Table 3or Table 4. In certain embodiments, a polypeptide includes a contiguousamino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a contiguous amino acid sequence selected from a sequence set forthin Table 3 or Table 4. In certain embodiments, a polypeptide includes anamino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or anyinteger within these numbers) contiguous amino acids of an amino acidsequence selected from a sequence set forth in Table 3 or Table 4.

In certain embodiments, the antibodies of the disclosure are encoded bya nucleotide sequence. Nucleotide sequences of the invention can beuseful for a number of applications, including: cloning, gene therapy,protein expression and purification, mutation introduction, DNAvaccination of a host in need thereof, antibody generation for, e.g.,passive immunization, PCR, primer and probe generation, and the like. Incertain embodiments, the nucleotide sequence of the invention comprises,consists of, or consists essentially of, a nucleotide sequence selectedfrom a sequence set forth in Table 3 or Table 4. In certain embodiments,a nucleotide sequence includes a nucleotide sequence at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from asequence set forth in Table 3 or Table 4. In certain embodiments, anucleotide sequence includes a contiguous nucleotide sequence at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotidesequence selected from a sequence set forth in Table 3 or Table 4. Incertain embodiments, a nucleotide sequence includes a nucleotidesequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integerwithin these numbers) contiguous nucleotides of a nucleotide sequenceselected from a sequence set forth in Table 3 or Table 4.

It will also be understood by one of ordinary skill in the art that theantibodies suitable for use in the methods disclosed herein may bealtered such that they vary in sequence from the naturally occurring ornative sequences from which they were derived, while retaining thedesirable activity of the native sequences. For example, nucleotide oramino acid substitutions leading to conservative substitutions orchanges at “non-essential” amino acid residues may be made. Mutationsmay be introduced by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis.

The antibodies suitable for use in the methods disclosed herein maycomprise conservative amino acid substitutions at one or more amino acidresidues, e.g., at essential or non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in a binding polypeptide is preferably replaced with anotheramino acid residue from the same side chain family. In certainembodiments, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members. Alternatively, in certain embodiments, mutations may beintroduced randomly along all or part of a coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be incorporatedinto binding polypeptides of the invention and screened for theirability to bind to the desired target.

As used herein, the term antigen “cross-presentation” refers topresentation of exogenous protein antigens to T cells via MHC class Iand class II molecules on APCs.

As used herein, the term “cross-reacts” refers to the ability of anantibody of the disclosure to bind to CD137 from a different species.For example, an antibody of the present disclosure which binds humanCD137 may also bind another species of CD137. As used herein,cross-reactivity is measured by detecting a specific reactivity withpurified antigen in binding assays (e.g., SPR, ELISA) or binding to, orotherwise functionally interacting with, cells physiologicallyexpressing CD137. Methods for determining cross-reactivity includestandard binding assays as described herein, for example, by Biacore™surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPRinstrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8+ T cells.

As used herein, the term “dimerization” refers to the formation of amacromolecular complex by two, usually non-covalently bound,macromolecules, such as proteins or multimers of proteins.Homodimerization refers to the process of dimerization when themacromolecules (e.g., proteins) are identical in nature.Heterodimerization refers to the process of dimerization when themacromolecules (e.g., proteins) are non-identical in nature. Methods fordetermining dimerization are known to those of skill in the art. Forexample, such methods include, but are not limited to, yeast two-hybridassay, fluorescence resonance energy transfer (FRET), bioluminescenceresonance energy transfer (BRET), protein mass spectrometry, evanescentwave methods, size exclusion chromatography, analyticalultracentrifugation, scattering techniques, NMR spectroscopy, isothermaltitration calorimetry, fluorescence anisotropy, fluorescence correlationspectroscopy (FCS), fluorescence recovery after photobleaching (FRAP),proximity imaging (PRIM) and bimolecular fluorescence complementation(BiFC) (see e.g., Gell D. A., Grant R. P., Mackay J. P. (2012) TheDetection and Quantitation of Protein Oligomerization. In: Matthews J.M. (eds) Protein Dimerization and Oligomerization in Biology. Advancesin Experimental Medicine and Biology, vol 747. Springer, New York, N.Y.;and Xie, Q. et al. Methods Mol Biol, 2011; 680: 3-28).

As used herein, the terms “dimerization of CD137” refers to thedimerization of two CD137 trimers. In some embodiments, the anti-CD137agonist antibodies described herein induce or enhance dimerization ofCD137. In some embodiments, the anti-CD137 agonist antibodies describedherein induce or enhance dimerization of CD137 relative to the amount ofdimerization in the absence of an anti-CD137 agonist antibody. In someembodiments, the anti-CD137 agonist antibodies described herein induceor enhance dimerization of CD137 relative to the amount of dimerizationin the presence of a reference anti-CD137 agonist antibody. In someembodiments, dimerization is increased by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100%.

As used herein, the term “EC₅₀” refers to the concentration of anantibody or an antigen-binding portion thereof, which induces aresponse, either in an in vitro or an in vivo assay, which is 50% of themaximal response, i.e., halfway between the maximal response and thebaseline.

As used herein, the term “effective dose” or “effective dosage” isdefined as an amount sufficient to achieve or at least partially achievethe desired effect. The term “therapeutically effective dose” is definedas an amount sufficient to cure or at least partially arrest the diseaseand its complications in a patient already suffering from the disease.Amounts effective for this use will depend upon the severity of thedisorder being treated and the general state of the patient's own immunesystem.

As used herein, the term “epitope” or “antigenic determinant” refers toa determinant or site on an antigen (e.g., CD137) to which anantigen-binding protein (e.g., an immunoglobulin, antibody, orantigen-binding fragment) specifically binds. The epitopes of proteinantigens can be demarcated into “linear epitopes” and “conformationalepitopes”. As used herein, the term “linear epitope” refers to anepitope formed from a contiguous, linear sequence of linked amino acids.Linear epitopes of protein antigens are typically retained upon exposureto chemical denaturants (e.g., acids, bases, solvents, cross-linkingreagents, chaotropic agents, disulfide bond reducing agents) or physicaldenaturants (e.g. thermal heat, radioactivity, or mechanical shear orstress). In some embodiments, an epitope is non-linear, also referred toas an interrupted epitope. As used herein, the term “conformationalepitope” or “non-linear epitope” refers to an epitope formed fromnoncontiguous amino acids juxtaposed by tertiary folding of apolypeptide. Conformational epitopes are typically lost upon treatmentwith denaturants. An epitope typically includes at least 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. In some embodiments, an epitope includes fewer than 25,24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6or 5 amino acids in a unique spatial conformation. Generally, anantibody, or antigen-binding fragment thereof, specific for a particulartarget molecule will preferentially recognize and bind to a specificepitope on the target molecule within a complex mixture of proteinsand/or macromolecules. In some embodiments, an epitope does not includeall amino acids of the extracellular domain of human CD137.

Also encompassed by the present disclosure are antibodies that bind toan epitope on CD137 which comprises all or a portion of an epitoperecognized by the particular antibodies described herein (e.g., the sameor an overlapping region or a region between or spanning the region).

As used herein, the term “epitope mapping” refers to a process or methodof identifying the binding site, or epitope, of an antibody, or antigenbinding fragment thereof, on its target protein antigen. Epitope mappingmethods and techniques are provided herein.

As used herein, the term “CD137” refers to a specific member of thetumor necrosis factor receptor (TNFR) family of transmembrane proteins.Alternative names and acronyms for CD137 in the art include “tumornecrosis factor receptor superfamily member 9” (TNFRSF9), 4-1BB and“induced by lymphocyte activation” (ILA) (Alderson et al., (1994) Eur JImmunol 24(9):2219-2227; Schwarz et al., (1993) Gene 134(2):295-298). Anexemplary amino acid sequence of full-length human CD137, includingleader, transmembrane, and cytoplasmic domains is set forth in Table 4(SEQ ID NO: 3) and here:

MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL.

As used herein, the term “CD137L” or “CD137 ligand” refers to a memberof the tumor necrosis factor (TNF) family of transmembrane proteins.Alternative names and acronyms for CD137L in the art include “tumornecrosis factor superfamily member 9” (TNFSF9) and 4-1BB ligand (4-1BBL)(Alderson et al., (1994) Eur J Immunol 24(9):2219-2227). An exemplaryamino acid sequence of full-length CD137L is set forth in Table 4 (SEQID NO: 97).

As used herein, the terms “Fc-mediated effector functions” or “Fceffector functions” refer to the biological activities of an antibodyother than the antibody's primary function and purpose. For example, theeffector functions of a therapeutic agnostic antibody are the biologicalactivities other than the activation of the target protein or pathway.Examples of antibody effect functions include C1q binding and complementdependent cytotoxicity; Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g., B cell receptor); lack of activation ofplatelets that express Fc receptor; and B cell activation. Many effectorfunctions begin with Fc binding to an Fcγ receptor.

As used herein, the term “Fc receptor” refers to a polypeptide found onthe surface of immune effector cells, which is bound by the Fc region ofan antibody. In some embodiments, the Fc receptor is an Fcγ receptor.There are three subclasses of Fcγ receptors, FcγRI (CD64), FcγRII (CD32)and FγcRIII (CD16). All four IgG isotypes (IgG1, IgG2, IgG3 and IgG4)bind and activate Fc receptors FcγRI, FcγRIIA and FcγRIIIA FcγRIIB is aninhibitory receptor, and therefore antibody binding to this receptordoes not activate complement and cellular responses. FcγRI is a highaffinity receptor that binds to IgG in monomeric form, whereas FcγRIIAand FcγRIIA are low affinity receptors that bind IgG only in multimericform and have slightly lower affinity. The binding of an antibody to anFc receptor and/or C1q is governed by specific residues or domainswithin the Fc regions. Binding also depends on residues located withinthe hinge region and within the CH2 portion of the antibody. In someembodiments, the agonistic and/or therapeutic activity of the antibodiesdescribed herein is dependent on binding of the Fc region to the Fcreceptor (e.g., FcγR). In some embodiments, the agonistic and/ortherapeutic activity of the antibodies described herein is enhanced bybinding of the Fc region to the Fc receptor (e.g., FcγR).

As used herein, the term “glycosylation pattern” is defined as thepattern of carbohydrate units that are covalently attached to a protein,more specifically to an immunoglobulin protein. A glycosylation patternof a heterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

As used herein, the term “hematological cancer” includes a lymphoma,leukemia, myeloma or a lymphoid malignancy, as well as a cancer of thespleen and lymph nodes. Exemplary lymphomas include both B celllymphomas (a B-cell hematological cancer) and T cell lymphomas. B-celllymphomas include both Hodgkin's lymphomas and most non-Hodgkin'slymphomas. Non-limiting examples of B cell lymphomas include diffuselarge B-cell lymphoma, follicular lymphoma, mucosa-associated lymphatictissue lymphoma, small cell lymphocytic lymphoma (overlaps with chroniclymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma,mediastinal large B cell lymphoma, Waldenstrom macroglobulinemia, nodalmarginal zone B cell lymphoma, splenic marginal zone lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,lymphomatoid granulomatosis. Non-limiting examples of T cell lymphomasinclude extranodal T cell lymphoma, cutaneous T cell lymphomas,anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma.Hematological malignancies also include leukemia, such as, but notlimited to, secondary leukemia, chronic lymphocytic leukemia, acutemyelogenous leukemia, chronic myelogenous leukemia, and acutelymphoblastic leukemia. Hematological malignancies further includemyelomas, such as, but not limited to, multiple myeloma and smolderingmultiple myeloma. Other hematological and/or B cell- orT-cell-associated cancers are encompassed by the term hematologicalmalignancy.

As used herein, the term “human antibody” includes antibodies havingvariable and constant regions (if present) of human germlineimmunoglobulin sequences. Human antibodies of the disclosure can includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo) (See, e.g., Lonberget al., (1994) Nature 368(6474): 856-859); Lonberg, (1994) Handbook ofExperimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern.Rev. Immunol. 13:65-93, and Harding & Lonberg, (1995) Ann. N.Y. Acad.Sci. 764:536-546). However, the term “human antibody” does not includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences (i.e. humanized antibodies).

As used herein, the term “heterologous antibody” is defined in relationto the transgenic non-human organism producing such an antibody. Thisterm refers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

The terms “inducing an immune response” and “enhancing an immuneresponse” are used interchangeably and refer to the stimulation of animmune response (i.e., either passive or adaptive) to a particularantigen. The term “induce” as used with respect to inducing CDC or ADCCrefer to the stimulation of particular direct cell killing mechanisms.

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment(such as treatment with a composition comprising an anti-CD137antibody).

The term “in vivo” refers to processes that occur in a living organism.

As used herein, the term “isolated antibody” is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to human CD137 is substantially free of antibodiesthat specifically bind antigens other than CD137). An isolated antibodythat specifically binds to an epitope may, however, havecross-reactivity to other CD137 proteins from different species.However, the antibody continues to display specific binding to humanCD137 in a specific binding assay as described herein. In addition, anisolated antibody is typically substantially free of other cellularmaterial and/or chemicals. In some embodiments, a combination of“isolated” antibodies having different CD137 specificities is combinedin a well-defined composition.

As used herein, the term “isolated nucleic acid molecule” refers tonucleic acids encoding antibodies or antibody portions (e.g., V_(H),V_(L), CDR3) that bind to CD137, is intended to refer to a nucleic acidmolecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind antigens other than CD137,which other sequences may naturally flank the nucleic acid in humangenomic DNA. For example, a sequence selected from a sequence set forthin Table 3 or Table 4 corresponds to the nucleotide sequences comprisingthe heavy chain (V_(H)) and light chain (V_(L)) variable regions ofanti-CD137 antibody monoclonal antibodies described herein.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In someembodiments, a human monoclonal antibody of the disclosure is of theIgG1 isotype. In some embodiments, a human monoclonal antibody of thedisclosure is of the IgG1 isotype and comprises a mutation. In someembodiments, a human monoclonal antibody of the disclosure is of theIgG2 isotype. In some embodiments, a human monoclonal antibody of thedisclosure is of the IgG3 isotype. In some embodiments, a humanmonoclonal antibody of the disclosure is of the IgG4 isotype. In someembodiments, a human monoclonal antibody of the disclosure is of theIgG4 isotype and comprises a mutation. In some embodiments, the mutationis a substitution at Ser228. In some embodiments, the substitution atSer228 is S228P.

As used herein, the term “isotype switching” refers to the phenomenon bywhich the class, or isotype, of an antibody changes from one Ig class toone of the other Ig classes.

As used herein the term “KD” or “K_(D)” refers to the equilibriumdissociation constant of a binding reaction between an antibody and anantigen. The value of K_(D) is a numeric representation of the ratio ofthe antibody off-rate constant (kd) to the antibody on-rate constant(ka). The value of K_(D) is inversely related to the binding affinity ofan antibody to an antigen. The smaller the K_(D) value the greater theaffinity of the antibody for its antigen. Affinity is the strength ofbinding of a single molecule to its ligand and is typically measured andreported by the equilibrium dissociation constant (K_(D)), which is usedto evaluate and rank order strengths of bimolecular interactions.

As used herein, the term “kd” or “k_(d)” (alternatively “koff” or“k_(off)”) is intended to refer to the off-rate constant for thedissociation of an antibody from an antibody/antigen complex. The valueof kd is a numeric representation of the fraction of complexes thatdecay or dissociate per second, and is expressed in units sec⁻¹.

As used herein, the term “ka” or “k_(a)” (alternatively “kon” or“k_(on)”) is intended to refer to the on-rate constant for theassociation of an antibody with an antigen. The value of ka is a numericrepresentation of the number of antibody/antigen complexes formed persecond in a 1 molar (1M) solution of antibody and antigen, and isexpressed in units M⁻¹ sec⁻¹.

As used herein, the terms “linked,” “fused”, or “fusion”, are usedinterchangeably. These terms refer to the joining together of two moreelements or components or domains, by whatever means including chemicalconjugation or recombinant means. Methods of chemical conjugation (e.g.,using heterobifunctional crosslinking agents) are known in the art.

As used herein, “local administration” or “local delivery,” refers todelivery that does not rely upon transport of the composition or agentto its intended target tissue or site via the vascular system. Forexample, the composition may be delivered by injection or implantationof the composition or agent or by injection or implantation of a devicecontaining the composition or agent. Following local administration inthe vicinity of a target tissue or site, the composition or agent, orone or more components thereof, may diffuse to the intended targettissue or site.

As used herein, “MHC molecules” refers to two types of molecules, MHCclass I and MHC class II. MHC class I molecules present antigen tospecific CD8+ T cells and MHC class II molecules present antigen tospecific CD4+ T cells. Antigens delivered exogenously to APCs areprocessed primarily for association with MHC class II. In contrast,antigens delivered endogenously to APCs are processed primarily forassociation with MHC class I.

As used herein, the term “monoclonal antibody” refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and optional constant regions derived from humangermline immunoglobulin sequences. In some embodiments, human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

As used herein, the term “multimerization” refers to the formation of amacromolecular complex comprising more than two macromolecules such asproteins, typically bound by non-covalent interactions. Methods fordetermining multimerization are known to those of skill in the art andare described supra for dimerization. In some embodiments, theanti-CD137 agonist antibodies described herein induce or enhancemultimerization of CD137. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance multimerization of CD137relative to the amount of multimerization in the absence of ananti-CD137 agonist antibody. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance multimerization of CD137relative to the amount of multimerization in the presence of a referenceanti-CD137 agonist antibody. In some embodiments, multimerization isincreased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

As used herein, the term “naturally-occurring” as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

As used herein, the term “nonswitched isotype” refers to the isotypicclass of heavy chain that is produced when no isotype switching hastaken place; the CH gene encoding the nonswitched isotype is typicallythe first CH gene immediately downstream from the functionallyrearranged VDJ gene. Isotype switching has been classified as classicalor non-classical isotype switching. Classical isotype switching occursby recombination events which involve at least one switch sequenceregion in the transgene. Non-classical isotype switching may occur by,for example, homologous recombination between human σ_(μ) and humanΣ_(μ) (δ-associated deletion). Alternative non-classical switchingmechanisms, such as intertransgene and/or interchromosomalrecombination, among others, may occur and effectuate isotype switching.

As used herein, the term “nucleic acid” refers to deoxyribonucleotidesor ribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides thathave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions can be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081, 1991; Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; andCassol et al, 1992; Rossolini et al, Mol. Cell. Probes 8:91-98, 1994).For arginine and leucine, modifications at the second base can also beconservative. The term nucleic acid is used interchangeably with gene,cDNA, and mRNA encoded by a gene.

Polynucleotides used herein can be composed of any polyribonucleotide orpolydeoxribonucleotide, which can be unmodified RNA or DNA or modifiedRNA or DNA. For example, polynucleotides can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that can be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the polynucleotide can be composed of triple-stranded regionscomprising RNA or DNA or both RNA and DNA. A polynucleotide can alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

As used herein, the term “paratope”, also “antigen-binding site” refersto a portion of an antibody, or antigen-binding fragment thereof, whichrecognizes and binds to an epitope on an antigen, comprising the set ofcomplementarity determining regions (CDRs) located within variable heavyand light chains.

As used herein, “parenteral administration,” “administeredparenterally,” and other grammatically equivalent phrases, refer tomodes of administration other than enteral and topical administration,usually by injection, and include, without limitation, intravenous,intranasal, intraocular, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural, intracerebral, intracranial,intracarotid and intrasternal injection and infusion.

As used herein, the term “patient” includes human and other mammaliansubjects that receive either prophylactic or therapeutic treatment.

The term “percent identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, the“percent identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared. For sequencecomparison, typically one sequence acts as a reference sequence to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

As generally used herein, “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

As used herein, a “pharmaceutically acceptable carrier” refers to, andincludes, any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Thecompositions can include a pharmaceutically acceptable salt, e.g., anacid addition salt or a base addition salt (see, e.g., Berge et al.(1977) J Pharm Sci 66:1-19).

As used herein, the terms “polypeptide,” “peptide”, and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

As used herein, the term “preventing” when used in relation to acondition, refers to administration of a composition which reduces thefrequency of, or delays the onset of, symptoms of a medical condition ina subject relative to a subject which does not receive the composition.

As used herein, the term “purified” or “isolated” as applied to any ofthe proteins (antibodies or fragments) described herein refers to apolypeptide that has been separated or purified from components (e.g.,proteins or other naturally-occurring biological or organic molecules)which naturally accompany it, e.g., other proteins, lipids, and nucleicacid in a prokaryote expressing the proteins. Typically, a polypeptideis purified when it constitutes at least 60 (e.g., at least 65, 70, 75,80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in asample.

As used herein, the term “rearranged” refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

As used herein, the term “receptor clustering” refers to a cellularprocess that results in grouping or local accumulation of a set ofreceptors at a particular cellular location, often to induce or amplifya signaling response. Many protein receptors bind cognate ligands andcluster, i.e., form dimers, trimers, oligomers or multimers, uponbinding their cognate ligands. For example, the PDGF receptor and TNFreceptor superfamily members form dimers and trimers upon ligandbinding, respectively. Cognate ligand-induced clustering (e.g.,dimerization, multimerization) induces signal transduction through thereceptor. Accordingly, the antibodies, or antigen-binding fragmentsthereof, of the present disclosure can activate a receptor by binding tomore than one receptor and induce or stabilize dimerization,trimerization, and/or multimerization with or without cognate ligandbinding.

Receptor clustering and multimerization is needed for TNFR signaling(Wajant (2015) Cell Death Differ 22(11):1727-1741), and in particularfor TNFRSF activation. 4-1BB (CD137), CD40, GITR, CD27, DR3, DRS, andFas are some of the TNFSF receptors known to require clustering in orderto trigger downstream signaling. Experimental evidence that the 4-1BBreceptor must be cross-linked to signal comes from Rabu et al. Theseauthors reported that a 1-trimer form of human 4-1BBL had no activatingeffects on human T cells whereas cross-linking the protein into 2- ormore trimers led to a strongly activating protein (Rabu et al., (2005) JBiol Chem 280:41472-41481). Accordingly, in some embodiments, ananti-CD137 agonist antibody induces the multimerization of 2 or moretrimers of CD137.

As used herein, the term “recombinant host cell” (or simply “host cell”)is intended to refer to a cell into which a recombinant expressionvector has been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell but to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “recombinant human antibody” includes all humanantibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences are encoded by thegermline genes, but include subsequent rearrangements and mutationswhich occur, for example, during antibody maturation. As known in theart (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid molecules that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not have sequence identity with the original nucleicacid molecules, but instead will be substantially identical or similar(i.e., have at least 80% identity).

As used herein, the term “reference antibody” (used interchangeably with“reference mAb”) or “reference antigen-binding protein” refers to anantibody, or an antigen-binding fragment thereof, that binds to aspecific epitope on human CD137 and is used to establish a relationshipbetween itself and one or more distinct antibodies. In some embodiments,the relationship is the binding of the reference antibody and the one ormore distinct antibodies to the same epitope on CD137. As used herein,the term connotes an anti-CD137 antibody that is useful in a test orassay, such as those described herein, (e.g., a competitive bindingassay), as a competitor, wherein the assay is useful for the discovery,identification or development, of one or more distinct antibodies thatbind to the same epitope. The variable heavy (V_(H)) and light chain(V_(L)) amino acid sequences of an exemplary reference antibody (mAb1)are provided in Table 4 (V_(H)1, SEQ ID NO. 4; V_(H)2, SEQ ID NO. 6). Insome embodiments, the term connotes an anti-CD137 antibody that isuseful in a test or assay, as a comparator, wherein the assay is usefulfor distinguishing characteristics of the antibodies (e.g.,hepatotoxicity, anti-tumor efficacy). In some embodiments, the referenceantibody is urelumab. In some embodiments, the reference antibody isutomilumab.

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody bindswith an equilibrium dissociation constant (K_(D)) of approximately lessthan 10⁻⁶ M, such as approximately less than 10⁻⁷, 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower when determined by surface plasmon resonance (SPR)technology in a BIACORE 2000 instrument using recombinant human CD137 asthe analyte and the antibody as the ligand and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The phrases “an antibody recognizing an antigen” and “anantibody specific for an antigen” are used interchangeably herein withthe term “an antibody which binds specifically to an antigen.”

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject with an immune disorder. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available at www dotgcg dot com), using a NWSgapdna. CMP matrix and a gap weight of 40, 50,60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percentidentity between two nucleotide or amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www dot gcg dot), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See www dot ncbi dot nlm dot nih dot gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present disclosure, while often ina native sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

As used herein, the term “tumor microenvironment” (alternatively “cancermicroenvironment”; abbreviated TME) refers to the cellular environmentor milieu in which the tumor or neoplasm exists, including surroundingblood vessels as well as non-cancerous cells including, but not limitedto, immune cells, fibroblasts, bone marrow-derived inflammatory cells,and lymphocytes. Signaling molecules and the extracellular matrix alsocomprise the TME. The tumor and the surrounding microenvironment areclosely related and interact constantly. Tumors can influence themicroenvironment by releasing extracellular signals, promoting tumorangiogenesis and inducing peripheral immune tolerance, while the immunecells in the microenvironment can affect the growth and evolution oftumor cells.

The term “T cell” refers to a type of white blood cell that can bedistinguished from other white blood cells by the presence of a T cellreceptor on the cell surface. There are several subsets of T cells,including, but not limited to, T helper cells (a.k.a. T_(H) cells orCD4⁺ T cells) and subtypes, including T_(H)1, T_(H)2, T_(H)3, T_(H)17,T_(H)9, and T_(FH) cells, cytotoxic T cells (i.e., Tc cells, CD8⁺ Tcells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memoryT cells and subtypes, including central memory T cells (T_(CM) cells),effector memory T cells (T_(EM) and T_(EMRA) cells), and resident memoryT cells (T_(RM) cells), regulatory T cells (a.k.a. T_(reg) cells orsuppressor T cells) and subtypes, including CD4⁺ FOXP3⁺ T_(reg) cells,CD4⁺FOXP3⁻ T_(reg) cells, Tr1 cells, Th3 cells, and T_(reg)17 cells,natural killer T cells (a.k.a. NKT cells), mucosal associated invariantT cells (MAITs), and gamma delta T cells (γδ T cells), including Vγ9/Vδ2T cells. Any one or more of the aforementioned or unmentioned T cellsmay be the target cell type for a method of use of the invention.

As used herein, the term “T cell activation” or “activation of T cells”refers to a cellular process in which mature T cells, which expressantigen-specific T cell receptors on their surfaces, recognize theircognate antigens and respond by entering the cell cycle, secretingcytokines or lytic enzymes, and initiating or becoming competent toperform cell-based effector functions. T cell activation requires atleast two signals to become fully activated. The first occurs afterengagement of the T cell antigen-specific receptor (TCR) by theantigen-major histocompatibility complex (MHC), and the second bysubsequent engagement of co-stimulatory molecules (e.g., CD28). Thesesignals are transmitted to the nucleus and result in clonal expansion ofT cells, upregulation of activation markers on the cell surface,differentiation into effector cells, induction of cytotoxicity orcytokine secretion, induction of apoptosis, or a combination thereof.

As used herein, the term “T cell-mediated response” refers to anyresponse mediated by T cells, including, but not limited to, effector Tcells (e.g., CD8⁺ cells) and helper T cells (e.g., CD4⁺ cells). T cellmediated responses include, for example, T cell cytotoxicity andproliferation.

As used herein, the terms “therapeutically effective amount” or“therapeutically effective dose,” or similar terms used herein areintended to mean an amount of an agent (e.g., an anti-CD137 antibody oran antigen-binding fragment thereof) that will elicit the desiredbiological or medical response (e.g., an improvement in one or moresymptoms of a cancer).

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of suchtreatment, a human antibody of the present disclosure, for example, asubject in need of an enhanced immune response against a particularantigen or a subject who ultimately may acquire such a disorder, inorder to prevent, cure, delay, reduce the severity of, or ameliorate oneor more symptoms of the disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

As used herein, the term “unrearranged” or “germline configuration”refers to the configuration wherein the V segment is not recombined soas to be immediately adjacent to a D or J segment.

As used herein, the term “vector” is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Preferred methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the presently disclosed methods and compositions. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Anti-CD137 Antibodies and Antigen-Binding Fragments Thereof

The present disclosure provides antibodies that specifically bind to andagonize CD137. In some aspects, the disclosure provides anti-CD137agonist antibodies that are useful for the treatment of cancer. In someembodiments, the anti-CD137 agonist antibodies induce cytokineproduction. In some embodiments, the anti-CD137 agonist antibodiesincrease the number of CD8+ T cells in the tumor microenvironment. Insome embodiments, the anti-CD137 agonist antibodies induce protectiveanti-tumor immunity. The disclosure also provides anti-CD137 agonistantibodies that, upon administration in vivo, do not substantiallyincrease intrasplenic or intrahepatic CD4+ and/or CD8+ T cellpopulations.

Human CD137 is a 255 amino acid transmembrane polypeptide (SEQ ID NO: 3;Accession No. NM_001561; NP_001552) and a member of thephylogenetically-conserved tumor necrosis factor receptor (TNFR)superfamily. CD137 (alternatively 4-1BB, TNFR superfamily 9) and itsligand (CD137L) are involved in the regulation of a wide range of immuneactivities. CD137 ligand cross-links its receptor, CD137, which isexpressed on activated T cells, and co-stimulates T cell activities.CD137 is an activation-induced co-stimulatory molecule. Recent studieshave revealed that CD137-mediated anti-cancer effects are largely basedon its ability to activate T cells, in particular, to induce a cytotoxicT lymphocyte (CTL) response, and induce cytokine production, inparticular, high amounts of IFNγ (Ye et al., (2014) Clin Cancer Res20(1):44-55). CD137 ligand is a transmembrane protein on the cellsurface and transmit signals into the cells on which it is expressed, aphenomenon referred to as “reverse signaling” or “back signaling”).CD137 ligand expression is found on most types of leukocytes and on somenonimmune cells. In monocytic cells (monocytes, macrophages, and DCs),CD137 ligand signaling induces activation, migration, survival, anddifferentiation.

Accordingly, in some embodiments, an isolated anti-CD137 agonistantibody, or antigen-binding fragment thereof, described herein, bindsto and agonizes CD137 and allows or promotes CD137L binding. In someembodiments, an isolated anti-CD137 agonist antibody, or antigen-bindingfragment thereof, described herein, binds to and agonizes CD137. In someembodiments, the anti-CD137 antibodies provided by the disclosure bindto and agonize CD137 and co-stimulate activation of T cells.

In some embodiments, an isolated anti-CD137 agonist antibody, orantigen-binding fragment thereof, described herein, has one or more ofthe following properties or characteristics:

a) specifically binds to human CD137;

b) binds to human and cynomolgus CD137; and

c) binds to human and mouse CD137.

In some embodiments, an anti-CD137 agonist antibody, or antigen-bindingfragment thereof, described herein, binds to CD137 and co-stimulates Tcell activities. In some embodiments, an anti-CD137 agonist antibody, orantigen-binding fragment thereof, described herein, binds to CD137 andinduces or enhances T cell activation, a cytotoxic T lymphocyte (CTL)response, T cell proliferation, cytokine production, or a combinationthereof. In some embodiments, an anti-CD137 agonist antibody, orantigen-binding fragment thereof, described herein, binds to CD137 andinduces or enhances T cell activation, a cytotoxic T lymphocyte (CTL)response, T cell proliferation, cytokine production, or a combinationthereof, in a tumor microenvironment. In some embodiments, an anti-CD137antibody, or antigen-binding fragment thereof, described herein, doesnot significantly induce or enhance intrahepatic and/or intrasplenic Tcell activation and/or T cell proliferation. In some embodiments, ananti-CD137 antibody, described herein, binds to CD137 and induces theproduction of IFNγ. In some embodiments, the antibodies provided by thedisclosure bind to CD137 and induce the production of IL-2, TNF-α,IL-13, or a combination thereof.

In some embodiments, the anti-CD137 antibodies described hereinspecifically bind to and agonize CD137. In some embodiments, agonism ofCD137 is measured by determining the concentration of cytokines producedby immune cells. Methods for analyzing cytokine production are known inthe art and utilized in the Examples. In some embodiments, an increasein cytokine production by immune cells indicates CD137 agonism. In someembodiments, agonism of CD137 is measured by analyzing T cellproliferation. In some embodiments, an increase in T cell proliferationindicates CD137 agonism. In some embodiments, agonism of CD137 ismeasured by measuring the level of cell signaling either throughquantitation of phosphorylation of relevant molecules or expression of agene reporter after a relevant promoter. In some embodiments, anincrease in cell signaling indicates CD137 agonism. In some embodiments,agonism of CD137 is measured by measuring the volume of a tumor. In someembodiments, a decrease in the volume of a tumor indicates CD137agonism.

In some embodiments, the anti-CD137 antibodies described herein induce,increase or stabilize oligomerization, multimerization, or other higherorder clustering of CD137. In some embodiments, the clustering of CD137on the cell surface is observed via fluorescence microscopy.

Provided herein are isolated monoclonal antibodies or antigen bindingfragments thereof, that bind to and agonize CD137. In some embodiments,the antibodies or antigen binding fragments thereof, (i) bind humanCD137 with an affinity (K_(D)) of about 30-100 nM (e.g., between about30 nM and about 100 nM); (ii) bind an epitope on human CD137 describedherein; and/or (iii) comprise a heavy chain CDR3 comprising the aminoacid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).

Affinity for CD137

In some embodiments, an isolated anti-CD137 agonist antibody, or antigenbinding fragment thereof, described herein, binds human CD137 with anaffinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM and about100 nM or between about 40 nM and about 100 nM). In some embodiments,the affinity of the anti-CD137 antibody to human CD137 is at least two(e.g., at least three, four, five, six, seven, eight, nine, or 10) foldhigher than the affinity of mAb10 for mouse CD137. In some embodiments,the affinity of the anti-CD137 antibody is no greater than 500, 450,400, 350, 300, 250, 200, 250, 200, 175, 150, 125, 110, or 100 nM. Insome embodiments, the affinity of the anti-CD137 antibody to human CD137is at least two (e.g., at least three, four, five, six, seven, eight,nine, or 10) fold higher than the affinity of mAb10 for mouse CD137, butno greater than 500, 450, 400, 350, 300, 250, 200, 250, 200, 175, 150,125, 110, or 100 nM. The affinity of the antibody is the strength ofbinding to a single CD137 polypeptide. In some embodiments, affinity isindicated by the equilibrium dissociation constant (K_(D)). The value ofK_(D) is inversely related to the binding affinity of an antibody to anantigen. Accordingly, the smaller the K_(D) value, the greater theaffinity of the antibody for its antigen.

Methods for determining the affinity of an antibody for its antigen areknown in the art. An exemplary method for determining binding affinityemploys surface plasmon resonance. Surface plasmon resonance is anoptical phenomenon that allows for the analysis of realtime biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51: 19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B.,et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnsson, B., et al.(1991) Anal. Biochem. 198:268-277.

In some embodiments, the anti-CD137 antibodies described herein bindhuman CD137 with an affinity (K_(D)) of about 30-100 nM (e.g., betweenabout 30 nM and about 100 nM). In some embodiments, the anti-CD137antibodies described herein bind human CD137 with an affinity (K_(D)) ofabout 40-100 nM. In some embodiments, the anti-CD137 antibodiesdescribed herein bind human CD137 with an affinity (K_(D)) of about30-40 nM, 40-50 nM, 50-60 nM, 60-70 nM, 70-80 nM, 80-90 nM, 90-100 nM,45-55 nM, 55-65 nM, 75-85 nM, 85-95 nM, 45-95 nM, 50-90 nM, 55-85 nM,60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM. In someembodiments, the anti-CD137 antibodies described herein bind human CD137with an affinity (K_(D)) of about 60-80 nM. In some embodiments, theanti-CD137 antibodies described herein bind human CD137 with an affinity(K_(D)) of about 60-75 nM.

In some embodiments, the anti-CD137 antibodies described herein bindhuman CD137 with an affinity (K_(D)) of about 60-90 nM. In someembodiments, the anti-CD137 antibodies described herein bind human CD137with an affinity (K_(D)) of about 50-80 nM. In some embodiments, theanti-CD137 antibodies described herein bind human CD137 with an affinity(K_(D)) of about 40-70 nM. In some embodiments, the anti-CD137antibodies described herein bind human CD137 with an affinity (K_(D)) ofabout 55-75 nM. In some embodiments, the anti-CD137 antibodies describedherein bind human CD137 with an affinity (K_(D)) of about 65-75 nM. Insome embodiments, the anti-CD137 antibodies described herein bind humanCD137 with an affinity (K_(D)) of about 60-80 nM. In some embodiments,the anti-CD137 antibodies described herein bind human CD137 with anaffinity (K_(D)) of about 55-85 nM. In some embodiments, the anti-CD137antibodies described herein bind human CD137 with an affinity (K_(D)) ofabout 50-90 nM. In some embodiments, the anti-CD137 antibodies describedherein bind human CD137 with an affinity (K_(D)) of about 45-95 nM. Insome embodiments, the anti-CD137 antibodies described herein bind humanCD137 with an affinity (K_(D)) of about 85-95 nM. In some embodiments,the anti-CD137 antibodies described herein bind human CD137 with anaffinity (K_(D)) of about 75-85 nM. In some embodiments, the anti-CD137antibodies described herein bind human CD137 with an affinity (K_(D)) ofabout 75-85 nM. In some embodiments, the anti-CD137 antibodies describedherein bind human CD137 with an affinity (K_(D)) of about 55-65 nM. Insome embodiments, the anti-CD137 antibodies described herein bind humanCD137 with an affinity (K_(D)) of about 45-55 nM. In some embodiments,the anti-CD137 antibodies described herein bind human CD137 with anaffinity (K_(D)) of about 80-90 nM. In some embodiments, the anti-CD137antibodies described herein bind human CD137 with an affinity (K_(D)) ofabout 70-80 nM. In some embodiments, the anti-CD137 antibodies describedherein bind human CD137 with an affinity (K_(D)) of about 60-70 nM. Insome embodiments, the anti-CD137 antibodies described herein bind humanCD137 with an affinity (K_(D)) of about 50-60 nM. In some embodiments,the anti-CD137 antibodies described herein bind human CD137 with anaffinity (K_(D)) of about 40-50 nM. In some embodiments, the anti-CD137antibodies described herein bind human CD137 with an affinity (K_(D)) ofabout 30-40 nM. In some embodiments, the anti-CD137 antibodies describedherein bind human CD137 with an affinity (K_(D)) of about 30 nM, about31 nM, about 32 nM, about 33 nM, about 34 nM, about 35 nM, about 36 nM,about 37 nM, about 38 nM, about 39 nM, about 40 nM, about 41 nM, about42 nM, about 43 nM, about 44 nM, about 45 nM, about 46 nM, about 47 nM,about 48 nM, about 49 nM, about 50 nM, about 51 nM, about 52 nM, about53 nM, about 54 nM, about 55 nM, about 56 nM, about 57 nM, about 58 nM,about 59 nM, about 60 nM, about 61 nM, about 62 nM, about 63 nM, about64 nM, about 65 nM, about 66 nM, about 67 nM, about 68 nM, about 69 nM,about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, about 80 nM,about 81 nM, about 82 nM, about 83 nM, about 84 nM, about 85 nM, about86 nM, about 87 nM, about 88 nM, about 89 nM, about 90 nM, about 91 nM,about 92 nM, about 93 nM, about 94 nM, about 95 nM, about 96 nM, about97 nM, about 98 nM, about 99 nM, about 100 nM, about 101 nM, about 102nM, about 103 nM, about 104 nM, about 105 nM, about 106 nM, about 107nM, about 108 nM, about 109 nM or about 110 nM.

In some embodiments, the anti-CD137 antibodies described herein bindhuman CD137 with an affinity (K_(D)) of at least 30 nM but less thanabout 110 nM, at least 31 nM but less than about 109 nM, at least 32 nMbut less than about 108 nM, at least 33 nM but less than about 107 nM,at least 34 nM but less than about 106 nM, at least 35 nM but less thanabout 105 nM, at least 36 nM but less than about 104 nM, at least 37 nMbut less than about 103 nM at least 38 nM but less than about 102 nM, atleast 39 nM but less than about 101 nM, at least 40 nM but less thanabout 100 nM; at least 41 nM but less than about 99 nM; least 42 nM butless than about 98 nM; least 43 nM but less than about 97 nM; at least44 nM but less than about 96 nM; at least 45 nM but less than about 95nM; at least 46 nM but less than about 94 nM; at least 47 nM but lessthan about 93 nM; at least 48 nM but less than about 92 nM; at least 49nM but less than about 91 nM; at least 50 nM but less than about 90 nM;at least 51 nM but less than about 89 nM; at least 52 nM but less thanabout 88 nM; at least 53 nM but less than about 87 nM; at least 54 nMbut less than about 86 nM; at least 55 nM but less than about 85 nM; atleast 56 nM but less than about 84 nM; at least 57 nM but less thanabout 83 nM; at least 58 nM but less than about 82 nM; at least 59 nMbut less than about 81 nM; at least 60 nM but less than about 80 nM; atleast 61 nM but less than about 79 nM; at least 62 nM but less thanabout 78 nM; at least 63 nM but less than about 77 nM; at least 64 nMbut less than about 76 nM; or at least 65 nM but less than about 75 nM.In some embodiments, the anti-CD137 antibodies described herein bindhuman CD137 with an affinity (K_(D)) of at least 40 nM but less thanabout 100 nM.

In some embodiments, the anti-CD137 antibodies described hereincross-react with CD137 polypeptides from more than one species. In someembodiments, the anti-CD137 antibodies described herein bind cynomolgusCD137 and human CD137. In some embodiments, the anti-CD137 antibodiesdescribed herein bind mouse CD137 and human CD137. In some embodiments,the anti-CD137 antibodies described herein bind human CD137, mouse CD137and cynomolgus CD137.

CD137 Epitope Binding

In some embodiments, the isolated monoclonal antibody, or antigenbinding portion thereof, that specifically binds to human CD137, bindsto an epitope on human CD137 comprising one or more (e.g., one, two,three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or all 25) of amino acids 111-132 of SEQID NO:3. In some embodiments, the isolated monoclonal antibody, orantigen binding portion thereof, that specifically binds to human CD137,binds to an epitope within amino acids 111-132 of SEQ ID NO:3. In someaspects, the disclosure provides an isolated monoclonal antibody, orantigen binding portion thereof, that specifically binds to human CD137,binds to all or a portion of amino acids 111-132 of SEQ ID NO:3. In someembodiments, an isolated anti-CD137 agonist antibody, or antigen bindingfragment thereof, described herein, binds to an epitope of human CD137comprising residue K114 of SEQ ID NO: 3. In some embodiments, anisolated anti-CD137 agonist antibody, or antigen binding fragmentthereof, described herein, binds to an epitope of human CD137 comprisingresidues E111, T113 and K114 of SEQ ID NO: 3. In some embodiments, anisolated anti-CD137 agonist antibody, or antigen binding fragmentthereof, described herein, binds to an epitope of human CD137 comprisingresidues E111, T113, K114, N126 and 1132 of SEQ ID NO: 3. In someembodiments, an isolated anti-CD137 agonist antibody, or antigen bindingfragment thereof, described herein, binds to an epitope of human CD137comprising E111, T113, K114, N126, I132 and P135 of SEQ ID NO: 3. Insome embodiments, an isolated anti-CD137 agonist antibody, or antigenbinding fragment thereof, described herein, binds to an epitope of humanCD137 comprising one or more residues E111, T113, K114, N126, I132 andP135 of SEQ ID NO: 3.

In some embodiments, an isolated anti-CD137 agonist antibody, or antigenbinding fragment thereof, described herein, binds to an epitope of humanCD137 comprising a sequence of one or more amino acid residuescorresponding to amino acid positions 100 to 135, 101 to 135, 102 to135, 103 to 135, 104 to 135, 105 to 135, 106 to 135, 107 to 135, 108 to135, 109 to 135, 110 to 135, or 111 to 135 of SEQ ID NO: 3. In someembodiments, an isolated anti-CD137 agonist antibody, or antigen bindingfragment thereof, described herein, binds to an epitope of human CD137comprising a sequence of one or more amino acid residues correspondingto amino acid positions 111 to 135 of SEQ ID NO: 3. In some embodiments,the epitope comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residuescorresponding to amino acid positions 111 to 135 of SEQ ID NO: 3.

In some embodiments, an isolated anti-CD137 agonist antibody, or antigenbinding fragment thereof, described herein, binds to an epitope of humanCD137 within amino acid positions 100 to 135, 101 to 135, 102 to 135,103 to 135, 104 to 135, 105 to 135, 106 to 135, 107 to 135, 108 to 135,109 to 135, 110 to 135, or 111 to 135 of SEQ ID NO: 3. In someembodiments, an isolated anti-CD137 agonist antibody, or antigen bindingfragment thereof, described herein, binds to an epitope of human CD137within amino acid positions 111 to 135 of SEQ ID NO: 3. In someembodiments, the epitope comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acidresidues corresponding to amino acid positions 111 to 135 of SEQ ID NO:3.

In some embodiments, an isolated anti-CD137 agonist antibody, or antigenbinding fragment thereof, described herein, binds to an epitope of humanCD137 comprising ELTK (corresponding to amino acid residues 111-114 ofSEQ ID NO: 3). In some embodiments, amino acid residue L112 can beanother amino acid residue.

In some embodiments, the epitope is a non-linear epitope. In someembodiments, mutation of amino acid residue K114 abrogates bindings ofan isolated anti-CD137 agonist antibody, or antigen binding fragmentthereof, described herein, to human CD137.

In some embodiments, isolated anti-CD137 agonist antibody, or antigenbinding fragment thereof, described herein, binds to an epitope of humanCD137 comprising a sequence of one or more amino acid residuescorresponding to amino acid positions 111 to 135 of SEQ ID NO: 3,wherein the epitope comprises at least amino acid K114, and wherein theantibody or antigen binding portion thereof binds mouse CD137 and doesnot bind rat CD137. In some embodiments, the epitope is a non-linearepitope. In some embodiments, the antibody or antigen binding portionthereof binds mouse CD137 and cynomolgus CD137 and does not bind ratCD137. In some embodiments, binding of an isolated anti-CD137 agonistantibody, or antigen binding fragment thereof, described herein, tohuman, mouse, rat and cynomolgus CD137 is determined by surface plasmonresonance (SPR).

In some embodiments, the antibody or antigen binding portion thereofbinds to mouse, cynomolgus or human CD137 with an affinity that is atleast 10, 20, 30, 40, 50, 100, 200, 500 or 1000 times greater than theantibody's affinity for rat CD137. In some embodiments, the antibody orantigen binding portion thereof binds to mouse, cynomolgus or humanCD137 with an affinity that is at least 10, 20, 30, 40, 50, 100, 200,500 or 1000 times greater than the antibody's affinity for a CD137polypeptide that does not comprise a lysine at position 114 relative tohuman CD137 of SEQ ID NO: 3.

In some embodiments, an isolated anti-CD137 agonist antibody, orantigen-binding fragment thereof, described herein, binds to an epitopeof human CD137 and competes with mAb1 for binding to the epitope ofhuman CD137. In some embodiments, an isolated anti-CD137 agonistantibody, or antigen-binding fragment thereof, described herein, bindsto and agonizes CD137. In some embodiments, the anti-CD137 antibodiesprovided by the disclosure bind to and agonize CD137 and co-stimulateactivation of T cells.

The present disclosure provides antibodies that compete for binding toan epitope on CD137 which comprises all or a portion of an epitoperecognized by one or more particular reference antibodies describedherein (e.g., mAb1). In some embodiments, the anti-CD137 antibodies bindto an epitope of human CD137 and compete with a reference antibody(e.g., mAb1) for binding to the epitope of human CD137 and wherein theantibody, or antigen binding fragment thereof, binds human CD137 with anequilibrium dissociation constant K_(D) of 1×10⁻⁶ or less. In someembodiments, the anti-CD137 antibodies bind to an epitope on CD137,wherein one or more mutations to the epitope inhibit, reduce, or blockbinding to both the antibodies and a reference antibody (e.g., mAb1). Insome embodiments, the reference antibody is the mAb1 antibody, describedherein. In some embodiments, the reference antibody is any one antibodyprovided in Table 3 or Table 4.

Accordingly, the anti-CD137 antibodies provided by the disclosure may beassessed through x-ray crystallographic analysis of a crystal structurecomprising an antibody bound to CD137, or a fragment or portion thereof.In some aspects, the epitopes that bound by the antibodies provided bythe disclosure are identified by determining the residues on the humanCD137 antigen that reside or are located within 4 angstroms (Å) of anantibody paratope residue, e.g., mAb1.

In some embodiments, the epitope bound by the anti-CD137 antibodiesdescribed herein is at least 3 amino acid residues. In some embodiments,the epitope bound by the anti-CD137 antibodies described herein is atleast 4 amino acid residues. In some embodiments, the epitope bound bythe anti-CD137 antibodies described herein is at least 5 amino acidresidues. In some embodiments, the epitope bound by the anti-CD137antibodies described herein is at least 6 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is at least 7 amino acid residues. In some embodiments, theepitope bound by the anti-CD137 antibodies described herein is at least8 amino acid residues. In some embodiments, the epitope bound by theanti-CD137 antibodies described herein is at least 9 amino acidresidues. In some embodiments, the epitope bound by the anti-CD137antibodies described herein is at least 10 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is at least 12 amino acid residues. In some embodiments, theepitope bound by the anti-CD137 antibodies described herein is at least3 amino acid residues. In some embodiments, the epitope bound by theanti-CD137 antibodies described herein is at least 13 amino acidresidues. In some embodiments, the epitope bound by the anti-CD137antibodies described herein is at least 14 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is at least 15 amino acid residues.

In some embodiments, the epitope bound by the anti-CD137 antibodiesdescribed herein is fewer than 25 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is fewer than 24 amino acid residues. In some embodiments, theepitope bound by the anti-CD137 antibodies described herein is fewerthan 23 amino acid residues. In some embodiments, the epitope bound bythe anti-CD137 antibodies described herein is fewer than 22 amino acidresidues. In some embodiments, the epitope bound by the anti-CD137antibodies described herein is fewer than 21 amino acid residues. Insome embodiments, the epitope bound by the anti-CD137 antibodiesdescribed herein is fewer than 20 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is fewer than 19 amino acid residues. In some embodiments, theepitope bound by the anti-CD137 antibodies described herein is fewerthan 18 amino acid residues. In some embodiments, the epitope bound bythe anti-CD137 antibodies described herein is fewer than 17 amino acidresidues. In some embodiments, the epitope bound by the anti-CD137antibodies described herein is fewer than 16 amino acid residues. Insome embodiments, the epitope bound by the anti-CD137 antibodiesdescribed herein is fewer than 15 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is fewer than 14 amino acid residues. In some embodiments, theepitope bound by the anti-CD137 antibodies described herein is fewerthan 13 amino acid residues. In some embodiments, the epitope bound bythe anti-CD137 antibodies described herein is fewer than 12 amino acidresidues. In some embodiments, the epitope bound by the anti-CD137antibodies described herein is fewer than 11 amino acid residues. Insome embodiments, the epitope bound by the anti-CD137 antibodiesdescribed herein is fewer than 10 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is fewer than 9 amino acid residues. In some embodiments, theepitope bound by the anti-CD137 antibodies described herein is fewerthan 8 amino acid residues. In some embodiments, the epitope bound bythe anti-CD137 antibodies described herein is fewer than 7 amino acidresidues. In some embodiments, the epitope bound by the anti-CD137antibodies described herein is fewer than 6 amino acid residues. In someembodiments, the epitope bound by the anti-CD137 antibodies describedherein is fewer than 5 amino acid residues.

In some embodiments, the anti-CD137 antibodies described herein bind toan epitope of fewer than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids and comprises amino acidresidue K114 of SEQ ID NO: 3.

Variable Regions

In some embodiments, provided herein are isolated monoclonal antibodiesor antigen binding fragments thereof, comprising heavy and light chainvariable sequences as set forth in Tables 3 and 4.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain CDRs selected from the group consisting of:

(a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 70, 79 and 90, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 71, 80 and 91, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 72, 81 and 92, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 73, 82 and 91, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 74, 83 and 93, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 75, 84 and 91, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 74, 85 and 94, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 76, 86 and 95, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 77, 87 and 93, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 88 and 90, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 57 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(m) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 58 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(n) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 59 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(o) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 60 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(p) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 61 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(q) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 58 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(r) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 62 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(s) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:52, 63 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(t) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 64 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(u) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 65 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(v) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 108 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(w) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:107, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively; and

(x) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 109, 110 and 92, respectively.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain variable regions, wherein the heavy chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 101and 103; and wherein the light chain variable region comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 6, 28,30, 32, 34, 36, 38, 40, 42, 44, 46 and 105.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain CDRs, wherein heavy chain CDR3 comprises the aminoacid sequence set forth in SEQ ID NO: 68.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain variable regions comprising amino acid sequencesselected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively;

(b) SEQ ID NO: 4 and 28, respectively;

(c) SEQ ID NO: 4 and 30, respectively;

(d) SEQ ID NO: 4 and 32, respectively;

(e) SEQ ID NO: 4 and 34, respectively;

(f) SEQ ID NO: 4 and 36, respectively;

(g) SEQ ID NO: 4 and 38, respectively;

(h) SEQ ID NO: 4 and 40, respectively;

(i) SEQ ID NO: 4 and 42, respectively;

(j) SEQ ID NO: 4 and 44, respectively;

(k) SEQ ID NO: 4 and 46, respectively;

(l) SEQ ID NO: 8 and 6, respectively;

(m) SEQ ID NO: 10 and 6, respectively;

(n) SEQ ID NO: 12 and 6, respectively;

(o) SEQ ID NO: 14 and 6, respectively;

(p) SEQ ID NO: 16 and 6, respectively;

(q) SEQ ID NO: 18 and 6, respectively;

(r) SEQ ID NO: 20 and 6, respectively;

(s) SEQ ID NO: 22 and 6, respectively;

(t) SEQ ID NO: 24 and 6, respectively;

(u) SEQ ID NO: 26 and 6, respectively;

(v) SEQ ID NO: 101 and 6, respectively;

(w) SEQ ID NO: 103 and 6, respectively; and

(x) SEQ ID NO: 4 and 105, respectively.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain variable regions, wherein the heavy chain variableregion comprises an amino acid sequence which is at least 90% identicalto the amino acid sequence selected from the group consisting of SEQ IDNOs: 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 101 and 103; and whereinthe light chain variable region comprises an amino acid sequence whichis at least 90% identical to the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 6, 28, 30, 32, 34, 36, 38, 40, 42, 44,46 and 105.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain variable regions comprising amino acid sequencesat least 90% identical to the amino acid sequences selected from thegroup consisting of:

(a) SEQ ID NO: 4 and 6, respectively;

(b) SEQ ID NO: 4 and 28, respectively;

(c) SEQ ID NO: 4 and 30, respectively;

(d) SEQ ID NO: 4 and 32, respectively;

(e) SEQ ID NO: 4 and 34, respectively;

(f) SEQ ID NO: 4 and 36, respectively;

(g) SEQ ID NO: 4 and 38, respectively;

(h) SEQ ID NO: 4 and 40, respectively;

(i) SEQ ID NO: 4 and 42, respectively;

(j) SEQ ID NO: 4 and 44, respectively;

(k) SEQ ID NO: 4 and 46, respectively;

(l) SEQ ID NO: 8 and 6, respectively;

(m) SEQ ID NO: 10 and 6, respectively;

(n) SEQ ID NO: 12 and 6, respectively;

(o) SEQ ID NO: 14 and 6, respectively;

(p) SEQ ID NO: 16 and 6, respectively;

(q) SEQ ID NO: 18 and 6, respectively;

(r) SEQ ID NO: 20 and 6, respectively;

(s) SEQ ID NO: 22 and 6, respectively;

(t) SEQ ID NO: 24 and 6, respectively;

(u) SEQ ID NO: 26 and 6, respectively;

(v) SEQ ID NO: 101 and 6, respectively;

(w) SEQ ID NO: 103 and 6, respectively; and

(x) SEQ ID NO: 4 and 105, respectively.

In some embodiments, provided herein are antibodies that specificallybind human CD137 comprising heavy chain and light chain variable regionsencoded by nucleotide sequences selected from the group consisting of:

(a) SEQ ID NO: 5 and 7, respectively;

(b) SEQ ID NO: 5 and 29, respectively;

(c) SEQ ID NO: 5 and 31, respectively;

(d) SEQ ID NO: 5 and 33, respectively;

(e) SEQ ID NO: 5 and 35, respectively;

(f) SEQ ID NO: 5 and 37, respectively;

(g) SEQ ID NO: 5 and 39, respectively;

(h) SEQ ID NO: 5 and 41, respectively;

(i) SEQ ID NO: 5 and 43, respectively;

(j) SEQ ID NO: 5 and 45, respectively;

(k) SEQ ID NO: 5 and 47, respectively;

(l) SEQ ID NO: 9 and 7, respectively;

(m) SEQ ID NO: 11 and 7, respectively;

(n) SEQ ID NO: 13 and 7, respectively;

(o) SEQ ID NO: 15 and 7, respectively;

(p) SEQ ID NO: 17 and 7, respectively;

(q) SEQ ID NO: 19 and 7, respectively;

(r) SEQ ID NO: 21 and 7, respectively;

(s) SEQ ID NO: 23 and 7, respectively;

(t) SEQ ID NO: 25 and 7, respectively;

(u) SEQ ID NO: 27 and 7, respectively;

(v) SEQ ID NO: 102 and 7, respectively;

(w) SEQ ID NO: 104 and 7, respectively; and

(x) SEQ ID NO: 5 and 106, respectively.

In some embodiments, provided herein are antibodies that specificallybind human CD137 comprising heavy chain and light chain variable regionsencoded by nucleotide sequences having at least 90% identity to thenucleotide sequences selected from the group consisting of:

(a) SEQ ID NO: 5 and 7, respectively;

(b) SEQ ID NO: 5 and 29, respectively;

(c) SEQ ID NO: 5 and 31, respectively;

(d) SEQ ID NO: 5 and 33, respectively;

(e) SEQ ID NO: 5 and 35, respectively;

(f) SEQ ID NO: 5 and 37, respectively;

(g) SEQ ID NO: 5 and 39, respectively;

(h) SEQ ID NO: 5 and 41, respectively;

(i) SEQ ID NO: 5 and 43, respectively;

(j) SEQ ID NO: 5 and 45, respectively;

(k) SEQ ID NO: 5 and 47, respectively;

(l) SEQ ID NO: 9 and 7, respectively;

(m) SEQ ID NO: 11 and 7, respectively;

(n) SEQ ID NO: 13 and 7, respectively;

(o) SEQ ID NO: 15 and 7, respectively;

(p) SEQ ID NO: 17 and 7, respectively;

(q) SEQ ID NO: 19 and 7, respectively;

(r) SEQ ID NO: 21 and 7, respectively;

(s) SEQ ID NO: 23 and 7, respectively;

(t) SEQ ID NO: 25 and 7, respectively;

(u) SEQ ID NO: 27 and 7, respectively;

(v) SEQ ID NO: 102 and 7, respectively;

(w) SEQ ID NO: 104 and 7, respectively; and

(x) SEQ ID NO: 5 and 106, respectively.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain variable regions, wherein the heavy chain variableregion is encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 102and 104; and wherein the light chain variable region is encoded by anucleotide sequence selected from the group consisting of SEQ ID NOs: 7,29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 106.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chain variable regions, wherein the heavy chain variableregion is encoded by a nucleotide sequence having at least 90% identityto a nucleotide sequence selected from the group consisting of SEQ IDNOs: 5, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 102 and 104; and whereinthe light chain variable region is encoded by a nucleotide sequencehaving at least 90% identity to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 7, 29, 31, 33, 35, 37, 39, 41, 43, 45,47 and 106.

In some embodiments, provided herein are anti-CD137 antibodies thatspecifically bind to human CD137 and comprise a heavy chain CDR3 havingthe amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X isany amino acid. In some embodiments, X is any amino acid except foralanine. In some embodiments, mutation of residues D95, L100, Y100E,Y100G, and/or Y100H of SEQ ID NO: 126, results in loss of binding tohuman CD137.

In some embodiments, provided herein are anti-CD137 antibodies thatspecifically bind to human CD137 and comprise a heavy chain CDR3 havingthe amino acid sequence DXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X isany amino acid. In some embodiments, mutation of residues F98, D100A,Y100D, and/or Y100F, and/or Y100H of SEQ ID NO: 126, to alanine resultsin loss of binding to human CD137. In some embodiments, mutation ofresidues F98, D100A, Y100D, and/or Y100F, and/or Y100H of SEQ ID NO:126, to any residue except for alanine, results in an increase inbinding to human CD137.

In some embodiments, provided herein are anti-CD137 antibodies thatspecifically bind to human CD137 and comprise a heavy chain CDR3 havingthe amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₁) (SEQ ID NO: 128),wherein X₁ is any amino acid, wherein X₂ is a non-polar amino acid,wherein X₃ is a non-polar amino acid, wherein X₄ is any amino acid,wherein X₅ is a polar amino acid, wherein X₆ is any amino acid, whereinX₇ is any amino acid, wherein X₈ is a polar amino acid, wherein X₉ is apolar amino acid, and wherein X₁₀ is any amino acid. In someembodiments, X₂ is proline, wherein X₃ is phenylalanine or tryptophan,wherein X₅ is aspartic acid or glutamic acid, wherein X₈ is tyrosine,and wherein X₉ is tyrosine.

The role of an amino acid residue within the heavy chain CDR3 of anantibody or antigen binding portion thereof, in binding to a specifiedtarget (e.g., CD137) can be determined by methods known to one of skillin the art. In some embodiments, an initial analysis using alaninescanning is completed to determine the critical residues for antigenbinding. As described herein, alanine scanning is a technique used todetermine the contribution of a specific wild-type residue to thestability or function(s) (e.g., binding affinity) of given protein orpolypeptide. The technique involves the substitution of an alanineresidue for a wild-type residue in a polypeptide, followed by anassessment of the stability or function(s) (e.g., binding affinity) ofthe alanine-substituted derivative or mutant polypeptide and comparisonto the wild-type polypeptide. In some embodiments, the residuesidentified as not critical are further evaluated to modulate the bindingof the antibody to the antigen (e.g., increase or decrease binding). Anon-limiting example of such analysis is deep mutational scanning. Thismethod allows for the evaluation of large numbers of mutations. In someembodiments, each amino acid residue within the heavy chain CDR3 ismutated to every amino acid residue (except for alanine), and binding isassessed. Other methods for analyzing the effect of amino acid residuemutations are known in the art. In some embodiments, these methods areutilized to assess the role of residues in all of the heavy chain andlight chain CDRs in binding to human CD137.

Exemplary CD137 Binding Antibodies

In some embodiments, the anti-CD137 antibodies described herein bindhuman CD137 with an affinity (K_(D)) of about 30-100 nM (e.g., betweenabout 30 nM and about 100 nM). In some embodiments, the anti-CD137antibodies described herein bind human CD137 with an affinity (K_(D)) ofabout 40-100 nM (e.g., between about 40 nM and about 100 nM). In someembodiments, the anti-CD137 antibodies described herein bind an epitopeon human CD137 described supra (e.g., comprising K114). In someembodiments, the anti-CD137 antibodies described herein comprise a heavychain CDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ IDNO: 126). In some embodiments, the anti-CD137 antibodies describedherein bind human CD137 with an affinity (K_(D)) of 30-100 nM (e.g.,between about 30 nM and about 100 nM) and bind an epitope on human CD137described supra (e.g., comprising K114). In some embodiments, theanti-CD137 antibodies described herein bind human CD137 with an affinity(K_(D)) of 30-100 nM (e.g., between about 30 nM and about 100 nM) andcomprise a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126). In some embodiments, the anti-CD137antibodies described herein bind an epitope on human CD137 describedsupra (e.g., comprising K114) and comprise a heavy chain CDR3 comprisingthe amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126). In someembodiments, the anti-CD137 antibodies described herein bind human CD137with an affinity (K_(D)) of 30-100 nM (e.g., between about 30 nM andabout 100 nM), bind an epitope on human CD137 described supra (e.g.,comprising K114), and comprise a heavy chain CDR3 comprising the aminoacid sequence DXXXXLXXXXYXYYX (SEQ ID NO: 126).

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM); and

(ii) comprise a heavy chain CDR3 comprising the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM); and

(ii) bind to an epitope on human CD137 comprising one or more residuesE111, T113, K114, N126, I132 and P135 of SEQ ID NO: 3.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope on human CD137 comprising one or more residuesE111, T113, K114, N126, I132 and P135 of SEQ ID NO: 3;

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid; or

(iv) combinations thereof.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope on human CD137 comprising one or more residuesE111, T113, K114, N126, I132 and P135 of SEQ ID NO: 3;

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid.; or

(iv) combinations thereof.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) specifically bind to an epitope on human CD137 comprising one ormore residues E111, T113, K114, N126, I132 and P135 of SEQ ID NO: 3; and

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope on human CD137 comprising one or more residuesE111, T113, K114, N126, I132 and P135 of SEQ ID NO: 3; and

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM); and

(ii) bind to an epitope comprising a sequence of one or more amino acidresidues corresponding to amino acid positions 111 to 135 of SEQ ID NO:3.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising a sequence of one or more amino acidresidues corresponding to amino acid positions 111 to 135 of SEQ ID NO:3;

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid; or

(iv) combinations thereof.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising a sequence of one or more amino acidresidues corresponding to amino acid positions 111 to 135 of SEQ ID NO:3;

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid; or

(iv) combinations thereof.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising a sequence of one or more amino acidresidues corresponding to amino acid positions 111 to 135 of SEQ ID NO:3; and

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising a sequence of one or more amino acidresidues corresponding to amino acid positions 111 to 135 of SEQ ID NO:3; and

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity of about 30-100 nM (e.g., betweenabout 30 nM and about 100 nM); and

(ii) bind to an epitope comprising ELTK (corresponding to amino acidresidues 111-114 of SEQ ID NO: 3).

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising ELTK (corresponding to amino acidresidues 111-114 of SEQ ID NO: 3);

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid; or

(iv) combinations thereof.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising ELTK (corresponding to amino acidresidues 111-114 of SEQ ID NO: 3);

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid; or

(iv) combinations thereof.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising ELTK (corresponding to amino acidresidues 111-114 of SEQ ID NO: 3); and

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid.

In some embodiments, the anti-CD137 antibodies

(i) bind human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM);

(ii) bind to an epitope comprising ELTK (corresponding to amino acidresidues 111-114 of SEQ ID NO: 3); and

(iii) comprise a heavy chain CDR3 comprising the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀(SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid.

In some embodiments, the anti-CD137 antibodies described supra compriseheavy and light chain CDRs selected from the group consisting of:

(a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively; and

(b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 108 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions, wherein the heavy chain variable regioncomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 4 and 101; and wherein the light chain variable regioncomprises an amino acid sequence of SEQ ID NO: 6.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions comprising amino acid sequences selected from thegroup consisting of:

(a) SEQ ID NOs: 4 and 6, respectively; and

(b) SEQ ID NOs: 101 and 6, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions comprising amino acid sequences selected from thegroup consisting of:

(a) SEQ ID NOs: 4 and 6, respectively;

(b) SEQ ID NOs: 101 and 6, respectively; and

(c) SEQ ID NOs: 26 and 6, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions encoded by nucleotide sequences selected from thegroup consisting of:

(a) SEQ ID NOs: 5 and 7, respectively; and

(b) SEQ ID NOs: 102 and 7, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions encoded by nucleotide sequences selected from thegroup consisting of:

(a) SEQ ID NOs: 5 and 7, respectively;

(b) SEQ ID NOs: 102 and 7, respectively; and

(c) SEQ ID NOs: 27 and 7, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions, wherein the heavy chain variable regioncomprises an amino acid sequence which is at least 90% identical to theamino acid sequence selected from the group consisting of SEQ ID NOs: 4and 101; and wherein the light chain variable region comprises an aminoacid sequence which is at least 90% identical to the amino acid sequenceof SEQ ID NO: 6.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions, wherein the heavy chain variable regioncomprises an amino acid sequence which is at least 90% identical to theamino acid sequence selected from the group consisting of SEQ ID NOs: 4,26 and 101; and wherein the light chain variable region comprises anamino acid sequence which is at least 90% identical to the amino acidsequence of SEQ ID NO: 6.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions, wherein the heavy chain variable region isencoded by a nucleotide sequence which is least 90% identical to thenucleotide sequence selected from the group consisting of SEQ ID NOs: 5and 102; and wherein the light chain variable region is encoded by anucleotide sequence which is at least 90% identical to the nucleotidesequence of SEQ ID NO: 7.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions, wherein the heavy chain variable region isencoded by a nucleotide sequence which is least 90% identical to thenucleotide sequence selected from the group consisting of SEQ ID NOs: 5,27 and 102; and wherein the light chain variable region is encoded by anucleotide sequence which is at least 90% identical to the nucleotidesequence of SEQ ID NO: 7.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions comprising amino acid sequences at least 90%identical to the amino acid sequences selected from the group consistingof:

(a) SEQ ID NOs: 4 and 6, respectively; and

(b) SEQ ID NOs: 101 and 6, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions comprising amino acid sequences at least 90%identical to the amino acid sequences selected from the group consistingof:

(a) SEQ ID NOs: 4 and 6, respectively;

(b) SEQ ID NOs: 101 and 6, respectively; and

(c) SEQ ID NOs: 26 and 6, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions encoded by nucleotide sequences at least 90%identical to the nucleotide sequences selected from the group consistingof:

(a) SEQ ID NOs: 5 and 7, respectively; and

(b) SEQ ID NOs: 102 and 7, respectively.

In some embodiments, the anti-CD137 antibodies comprise heavy and lightchain variable regions encoded by nucleotide sequences at least 90%identical to the nucleotide sequences selected from the group consistingof:

(a) SEQ ID NOs: 5 and 7, respectively;

(b) SEQ ID NOs: 102 and 7, respectively; and

(c) SEQ ID NOs: 27 and 7, respectively.

In some embodiments, the anti-CD137 antibodies described herein have atleast the functional properties of mAb1 (i.e., an antibody comprisingthe heavy and light chain variable sequences of SEQ ID NOs: 4 and 6,respectively), mab8 (i.e., an antibody comprising the heavy and lightchain variable sequences of SEQ ID NOs: 101 and 6, respectively) ormAb10 (i.e., an antibody comprising the heavy and light chain variablesequences of SEQ ID NOs: 26 and 6, respectively). In some embodiments,the functional properties of an antibody described herein include butare not limited to: induction or enhancement of dimerization of CD137;induction or enhancement of multimerization of CD137; induction orenhancement of CD137-mediated T cell activation; induction orenhancement of CD137-mediated cytotoxic T cell response; induction orenhancement of CD137-mediated T cell proliferation; induction orenhancement of CD137-mediated cytokine production; lack of induction orenhancement of intrahepatic and/or intrasplenic T cell activation and/orT cell proliferation; and reduction or inhibition of tumor growth.

In some embodiments, the anti-CD137 antibodies described herein bindhuman CD137 with an equilibrium dissociation constant K_(D) at leastequivalent to that of mAb1 (i.e., an antibody comprising the heavy andlight chain variable sequences of SEQ ID NOs: 4 and 6, respectively),mab8 (i.e., an antibody comprising the heavy and light chain variablesequences of SEQ ID NOs: 101 and 6, respectively) or mAb10 (i.e., anantibody comprising the heavy and light chain variable sequences of SEQID NOs: 26 and 6, respectively).

In some embodiments, the anti-CD137 antibodies described herein comprisea human IgG1 heavy chain constant region or a human IgG4 heavy chainconstant region. In some embodiments, the anti-CD137 antibodiesdescribed herein comprise a human wild-type IgG1 heavy chain constantregion or a human wild-type IgG4 heavy chain constant region. In someembodiments, the anti-CD137 antibodies described herein comprise a humanwild-type IgG1 heavy chain constant region as set forth in SEQ ID NO: 1.In some embodiments, the anti-CD137 antibodies described herein comprisea mutant IgG1 heavy chain constant region or a mutant IgG4 heavy chainconstant region. In some embodiments, the anti-CD137 antibodiesdescribed herein comprise a mutant IgG4 heavy chain constant region,wherein the mutant IgG4 heavy chain constant region comprises an aminoacid substitution at residue Ser228. In some embodiments, the amino acidsubstitution at residue Ser228 is S228P. In some embodiments, theanti-CD137 antibodies described herein comprise an IgG4 heavy chainconstant region, wherein the c-terminal lysine residue is removed. Insome embodiments, the anti-CD137 antibodies described herein comprise anIgG4 heavy chain constant region wherein the c-terminal lysine residueis removed and comprises the S228P amino acid substitution. In someembodiments, the anti-CD137 antibodies described herein comprise an IgG4heavy chain constant region as set forth in SEQ ID NO: 2.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chains comprising the amino acid sequences set forth inSEQ ID NOs: 129 and 133, respectively. In some embodiments, theanti-CD137 antibodies described herein comprise heavy and light chainscomprising the amino acid sequences set forth in SEQ ID NOs: 130 and133, respectively. In some embodiments, the anti-CD137 antibodiesdescribed herein comprise heavy and light chains comprising the aminoacid sequences set forth in SEQ ID NOs: 131 and 133, respectively. Insome embodiments, the anti-CD137 antibodies described herein compriseheavy and light chains comprising the amino acid sequences set forth inSEQ ID NOs: 132 and 133, respectively.

In some embodiments, the anti-CD137 antibodies described herein compriseheavy and light chains comprising amino acid sequences having at least80%, at least 85%, at least 90%, at least 95%, at least, 96%, at least97%, at least 98%, or at least 99% identity to SEQ ID NOs: 129 and 133,respectively. In some embodiments, the anti-CD137 antibodies describedherein comprise heavy and light chains comprising amino acid sequenceshaving at least 80%, at least 85%, at least 90%, at least 95%, at least,96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs:130 and 133, respectively. In some embodiments, the anti-CD137antibodies described herein comprise heavy and light chains comprisingamino acid sequences having at least 80%, at least 85%, at least 90%, atleast 95%, at least, 96%, at least 97%, at least 98%, or at least 99%identity to SEQ ID NOs: 131 and 133, respectively. In some embodiments,the anti-CD137 antibodies described herein comprise heavy and lightchains comprising amino acid sequences having at least 80%, at least85%, at least 90%, at least 95%, at least, 96%, at least 97%, at least98%, or at least 99% identity to SEQ ID NOs: 132 and 133, respectively.

Characterization and Functions of CD137 Binding Antibodies

I. Affinity

In some embodiments, an anti-CD137 antibody described herein binds humanCD137 with an affinity (KD) of about 40-100 nM (e.g., between about 40nM and about 100 nM) as determined by an antigen-binding assay. In someembodiments, an anti-CD137 antibody described herein binds human CD137with an affinity (KD) of about 30-100 nM (e.g., between about 30 nM andabout 100 nM) as determined by an antigen-binding assay. In someembodiments, an anti-CD137 antibody described herein binds human CD137with an affinity (KD) of about 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM,65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90 nM as determined by anantigen-binding assay.

In some embodiments, the antigen-binding assay determines a bindingaffinity of the anti-CD137 antibody for a CD137 polypeptide. In someembodiments, the antigen binding assay is surface plasmon resonance.Accordingly, in some embodiments an anti-CD137 antibody described hereinbinds human CD137 with an affinity (KD) of about 40-100 nM (e.g.,between about 40 nM and about 100 nM) as determined using surfaceplasmon resonance. In some embodiments, an anti-CD137 antibody describedherein binds human CD137 with an affinity (KD) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM) as determined using surfaceplasmon resonance. In some embodiments, an anti-CD137 antibody describedherein binds human CD137 with an affinity (KD) of about 45-95 nM, 50-90nM, 55-85 nM, 60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM, 50-80 nM, or 60-90nM as determined using surface plasmon resonance.

The phrase “surface plasmon resonance” includes an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore system (Pharmacia Biosensor AB,Uppsala, Sweden and Piscataway, N.J.). For further descriptions, seeJonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., etal. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.198:268-277. In some embodiments, the antigen binding assay is biolayerinterferometry (BLI). Accordingly, in some embodiments an anti-CD137antibody described herein binds human CD137 with an affinity (KD) ofabout 40-100 nM (e.g., between about 40 nM and about 100 nM) asdetermined using biolayer interferometry. In some embodiments, ananti-CD137 antibody described herein binds human CD137 with an affinity(KD) of about 30-100 nM (e.g., between about 30 nM and about 100 nM) asdetermined using biolayer interferometry. In some embodiments, ananti-CD137 antibody described herein binds human CD137 with an affinity(KD) of about 45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM, 55-75nM, 40-70 nM, 50-80 nM, or 60-90 nM as determined using biolayerinterferometry.

The phrase “biolayer interferometry” or “BLI” includes an opticalphenomenon that allows for the measurement of sub-nanometer changes inthe thickness of its optical layer detection surface. In someembodiments, biomolecules binds at a sensor surface and change theoptical layer thickness. The magnitude of the optical layer thicknesschange is proportional to the mass or molecular weight of the bindingmolecule. In some embodiments, CD137 is immobilized to the sensorsurface to measure binding by an antibody, wherein binding creates achanges in the molecular weight to produce a corresponding change in theoptical layer thickness. In some embodiments, BLI is performed with anOCTET system (ForteBio).

II. Immune Cell Effects

In some embodiments, an anti-CD137 antibody described herein induces orenhances cytokine production by an immune cell as determined by acytokine assay. In some embodiments, the cytokine assay determines anamount of at least one cytokine secreted from an immune cell contactedwith the anti-CD137 antibody, wherein an increase in the amount of theat least one cytokine indicates induction or enhancement of cytokineproduction by the anti-CD137 antibody. In some embodiments, an increasein cytokine production is at least 1 fold, 2 fold, 3 fold, 4 fold or 5fold more compared to a control antibody (e.g., an antibody that doesnot bind to CD137 and does not induce cytokine production).

In some embodiments, an anti-CD137 antibody described herein induces orenhances cytokine production by an immune cell as determined by acytokine assay, wherein the cytokine assay comprises the followingsteps:

(i) contacting the immune cell with the anti-CD137 antibody; and

(ii) determining an amount of at least one cytokine produced by theimmune cell,

wherein an increase in the amount of the at least one cytokine indicatesthe anti-CD137 antibody induces or enhances cytokine production by theimmune cell.

In some embodiments, an anti-CD137 antibody described herein induces orenhances cytokine production by an immune cell as determined by acytokine assay, wherein the cytokine assay comprises the followingsteps:

(i) contacting the immune cell with an anti-CD137 antibody; and

(ii) determining an amount of at least one cytokine produced by theimmune cell, and

(iii) comparing the amount of the at least one cytokine produced by theimmune cell to an amount secreted from a reference immune cell,

wherein the reference immune cell is contacted with a control antibody,and wherein an increase in the amount of the at least one cytokineproduced from the immune cell relative to the reference immune cellindicates induction or enhancement of human CD137-mediated cytokineproduction.

In some embodiments, an anti-CD137 antibody described herein induces orenhances cytokine production by an immune cell as determined by acytokine assay, wherein the cytokine assay comprises the followingsteps:

(i) contacting an immune cell with an anti-CD137 antibody;

(ii) determining an amount of at least one cytokine produced by theimmune cell, and

(iii) comparing the amount of the at least one cytokine produced by theimmune cell to an amount or level secreted from a reference immune cell,

wherein the reference immune cell is not contacted with the anti-CD137antibody, and wherein an increase in the amount of the at least onecytokine produced from the immune cell relative to the reference immunecell indicates induction or enhancement of human CD137-mediated cytokineproduction by the immune cell.

In some embodiments, the at least one cytokine is selected from a groupconsisting of IL-2, IFNγ, TNFα, IL-13, and combinations thereof. In someembodiments, the cytokine is IL-2. In some embodiments, the cytokine isIFNγ. In some embodiments, the cytokine is TNFα. In some embodiments,the cytokine is IL-13. In some embodiments, an anti-CD137 antibodyinduces or enhances IL-2 production. In some embodiments, an anti-CD137antibody induces or enhances TNFα production. In some embodiments, ananti-CD137 antibody induces or enhances IL-13 production. In someaspects, the cytokine produced is IL-2. In some aspects, the cytokineproduced is TNFα. In some aspects, the cytokine produced is IL-13. Insome aspects, the cytokine produced is IFNγ. In some aspects, thecytokine produced is IL-2 and TNFα. In some aspects, the cytokineproduced is IL-2 and IL-13. In some aspects, the cytokine produced isIL-2 and IFNγ. In some aspects, the cytokine produced is TNFα and IL-13.In some aspects, the cytokine produced is TNFα and IFNγ. In someaspects, the cytokine produced is IL-13 and IFNγ. In some aspects, thecytokine produced is IL-2, TNFα and IL-13. In some aspects, the cytokineproduced is IL-2, TNFα and IFNγ. In some aspects, the cytokine producedis IFNγ, TNFα and IL-13.

In some embodiments, the immune cell is a T cell. In some embodiments,the reference immune cell is a T cell. In some embodiments the T cell isa CD8+ T cell.

In some embodiments, the cytokine assay is a cytokine bead array assay.A cytokine bead array assay is a bead-based immunoassay that allows formultianalyte flow cytometric determination of multiple cytokines in asample. The use of microspheres of different size or color is the basisof a cytokine bead array assay, wherein each microsphere (or “bead”) iscoated with an antibody that specifically binds to an antigen (e.g., acytokine). Antibody-coated beads are then introduced to a sample incombination with detector antibodies. The bead:antigen:detector antibodycomplexes are then analyzed by flow cytometry. Commercially availablecytokine bead array assays include, but are not limited to, BD™Cytometric Bead Array Systems (BD Biosciences) and Luminex® Assays (R&DSystems). In some embodiments, induction or enhancement of humanCD137-mediated cytokine production is determined by a cytokine beadarray assay. In some embodiments, induction or enhancement of humanCD137-mediated cytokine production is determined by a Luminex® Assay.

In some embodiments, the cytokine assay is a Meso Scale Discovery (MSD)assay (Meso Scale Diagnostics; Rockville, Md.). An MSD assay is acommercially available assay based on detection ofelectrochemiluminescent-labeled antibodies that specifically bind to anantigen (e.g., a cytokine) of interest. An MSD assay comprises highbinding carbon electrodes in the bottom of microplate wells that allowfor attachment of biological reagents (e.g., capture antibodies specificfor a cytokine). MSD assays use electrochemiluminescent labels that areconjugated to detection antibodies. A sample is added to the microplatewells and electricity is applied to the plate electrodes by an MSDinstrument leading to light emission by the electrochemiluminescentlabels. Light intensity is measured to quantify analytes (e.g.,cytokines) in the sample. In some embodiments, induction or enhancementof human CD137-mediated cytokine production is determined by a MesoScale Discovery (MSD) assay.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay.In some embodiments, the T cell activation assay determines an amount ofat least one cytokine secreted from T cells contacted with an anti-CD137antibody described herein, wherein an increase in the amount of the atleast one cytokine indicates induction or enhancement of T cellactivation. In some embodiments, an increase in cytokine production isat least 1 fold, 2 fold, 3 fold, 4 fold or 5 fold more compared to acontrol antibody (e.g., an antibody that does not bind to CD137 and doesnot induce cytokine production).

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises the following steps:

(i) isolating T cells from a subject;

(ii) contacting the T cells with an anti-CD137 antibody; and

(iii) determining an amount of at least one cytokine secreted by the Tcells after (ii),

wherein an increase in the level of the at least one cytokine indicatesthe anti-CD137 antibody induces or enhances T cell activation.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises the following steps:

(i) isolating T cells from a subject;

(ii) contacting the T cells with an anti-CD137 antibody;

(iii) determining an amount of at least one cytokine secreted by the Tcells; and

(iv) comparing the amount of the at least one cytokines produced by theT cells to an amount or level secreted from reference T cells,

wherein the reference T cells are not contacted with the anti-CD137antibody, and wherein an increase in the amount of the at least onecytokine produced from the T cells relative to the reference T cellsindicates the anti-CD137 antibody induces or enhances T cell activation.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises the following steps:

(i) isolating T cells from a subject;

(ii) contacting the T cells with an anti-CD137 antibody;

(iii) determining an amount of at least one cytokine secreted by the Tcells; and

(iv) comparing the amount of the at least one cytokine produced by the Tcells to an amount secreted from reference T cells,

wherein the reference T cells are contacted with a control antibody, andwherein an increase in the amount of the at least one cytokine producedfrom the T cells relative to the reference T cells indicates theanti-CD137 antibody induces or enhances T cell activation.

In some embodiments, the T cell activation assay comprises determiningthe level of at least one cytokine secreted by the T cells after contactwith an anti-CD137 antibody described herein, wherein the at least onecytokine is selected from the group consisting of IL-2, IFNγ, TNFα andIL-13. In some embodiments, the cytokine is IL-2. In some embodiments,the cytokine is IFNγ. In some embodiments, the cytokine is TNFα. In someembodiments, the cytokine is IL-13. In some embodiments, the T cellactivation assay comprises a cytokine assay, such as those describedherein, to determine the amount of the at least one cytokine. In someaspects, the cytokine produced is IL-2. In some aspects, the cytokineproduced is TNFα. In some aspects, the cytokine produced is IL-13. Insome aspects, the cytokine produced is IFNγ. In some aspects, thecytokine produced is IL-2 and TNFα. In some aspects, the cytokineproduced is IL-2 and IL-13. In some aspects, the cytokine produced isIL-2 and IFNγ. In some aspects, the cytokine produced is TNFα and IL-13.In some aspects, the cytokine produced is TNFα and IFNγ. In someaspects, the cytokine produced is IL-13 and IFNγ. In some aspects, thecytokine produced is IL-2, TNFα and IL-13. In some aspects, the cytokineproduced is IL-2, TNFα and IFNγ. In some aspects, the cytokine producedis IFNγ, TNFα and IL-13.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises detecting surfaceexpression of at least one activation marker on T cells, and wherein anincrease in the expression level of the at least one activation markerindicates induction or enhancement of T cell activation. In someembodiments, “increase in surface expression” refers to at least a 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% increase in surface expression relative tosurface expression in the presence of a control antibody or in theabsence of an antibody.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay invitro, wherein the T cell activation assay comprises the followingsteps:

(i) isolating T cells from a subject;

(ii) contacting the T cells with an anti-CD137 antibody; and

(iii) detecting surface expression of at least one activation marker onthe T cells,

wherein an increase in surface expression of at least one activationmarker indicates the anti-CD137 antibody induces or enhances T cellactivation.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises the following steps:

(i) isolating T cells from a subject;

(ii) contacting the T cells with an anti-CD137 antibody;

(iii) determining surface expression of at least one activation markeron the T cells; and

(iv) comparing surface expression of at least one activation marker onthe T cells to surface expression of the at least one activation markeron reference T cells,

wherein the reference T cells are not contacted with the anti-CD137antibody, and wherein an increase in surface expression of at least oneactivation marker on the T cells relative to the reference T cellsindicates the anti-CD137 antibody induces or enhances T cell activation.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises the following steps:

(i) isolating T cells from a subject;

(ii) contacting the T cells with an anti-CD137 antibody;

(iii) determining surface expression of at least one activation markeron the T cells,

(iv) comparing the surface expression of the at least one activationmarker on the T cells to surface expression of the at least oneactivation marker on reference T cells,

wherein the reference T cells are contacted with a control antibody, andwherein an increase in surface expression of the at least one activationmarker on the T cells relative to surface expression of the at least oneactivation marker on the reference T cells indicates the anti-CD137antibody induces or enhances T cell activation.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay invivo, wherein the T cell activation assay comprises the following steps:

(i) administering the anti-CD137 antibody to a subject;

(ii) isolating T cells from the subject; and

(iii) detecting surface expression of at least one activation marker onthe T cells,

wherein an increase in surface expression of at least one activationmarker indicates the anti-CD137 antibody induces or enhancesCD137-mediated T cell activation.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises the following steps:

(i) administering the anti-CD137 antibody to a subject;

(ii) isolating T cells from the subject;

(iii) determining surface expression of at least one activation markeron the T cells after; and

(iv) comparing surface expression of the at least one activation markeron the T cells to surface expression of the at least one activationmarker on reference T cells,

wherein the reference T cells are isolated from a subject notadministered the anti-CD137 antibody, and wherein an increase in surfaceexpression of the at least one activation marker on the T cells relativeto the reference T cells indicates the anti-CD137 antibody induces orenhances T cell activation.

In some embodiments, an anti-CD137 antibody described herein induces orenhances T cell activation as determined by a T cell activation assay,wherein the T cell activation assay comprises the following steps:

(i) administering the anti-CD137 antibody to a subject;

(ii) isolating T cells from the subject;

(iii) determining surface expression of at least one activation markeron the T cells; and

(iv) comparing surface expression of the at least one activation markeron the T cells to surface expression of the at least one activationmarker on reference T cells,

wherein the reference T cells are isolated from a subject contacted witha control antibody, and wherein an increase in surface expression of theat least one activation marker on the T cells relative to surfaceexpression of the at least one activation marker on the reference Tcells indicates the anti-CD137 antibody induces or enhances T cellactivation.

In some embodiments, an anti-CD137 antibody described herein does notinduce or enhance intrahepatic T cell activation as determined by a Tcell activation assay in vivo, wherein the T cell activation assaycomprises the following steps:

(i) administering the anti-CD137 to a subject;

(ii) isolating T cells from the liver of the subject;

(iii) detecting surface expression of at least one activation marker onthe T cells; and

(iv) comparing the surface expression of the at least one activationmarker on the T cells to surface expression of the at least oneactivation marker on reference T cells,

wherein the reference T cells are isolated from a subject notadministered the anti-CD137 antibody, optionally, wherein the referenceT cells are isolated from a subject administered a control antibody, andwherein an absence of an increase in surface expression of the at leastone activation marker on the T cells relative to surface expression ofthe at least one activation marker on the reference T cells indicatesthe anti-CD137 antibody induces or enhances T cell activation.

In some embodiments, an anti-CD137 antibody described herein does notinduce or enhance intrasplenic T cell activation as determined by a Tcell activation assay in vivo, wherein the T cell activation assaycomprises the following steps:

(i) administering the anti-CD137 to a subject;

(ii) isolating T cells from the spleen of the subject;

(iii) detecting surface expression of at least one activation marker onthe T cells; and

(iv) comparing surface expression of the at least one activation markeron the T cells to surface expression of the at least one activationmarker on reference T cells,

wherein the reference T cells are isolated from a subject notadministered the anti-CD137 antibody, optionally, wherein the referenceT cells are isolated from a subject administered a control antibody, andwherein an absence in an increase in surface expression of the at leastone activation marker on the T cells relative to surface expression ofthe at least one activation marker on the reference T cells indicatesthe anti-CD137 antibody induces or enhances T cell activation.

In some embodiments “does not induce or enhance” is intended to refer tothe absence of an activity (e.g., T cell activation) or a lack ofincrease of an activity relative to an increase by a reference antibody.

In some embodiments, a surface expression of a T cell activation markeris equivalent to the surface expression in the absence of an antibody.In some embodiments a surface expression of a T cell activation markeris less than the surface expression in the presence of a referenceantibody that induces or enhance surface expression at least 1 fold, 5fold, 10 fold, 50 fold, or 100 fold higher compared to surfaceexpression in the absence of an antibody.

In some embodiments, the at least one activation marker is selected fromthe group consisting of CD25, CD69 and CD40L. In some embodiments, theone or more activation markers is CD25.

In some embodiments, T cells are isolated from a subject having a tumor.In some embodiments, the T cells are isolated from the tumor. In someembodiments, the control antibody is an isotype control antibody.

In some embodiments, an anti-CD137 antibody described herein induces orenhances infiltration of one or more immune cells into a tumormicroenvironment as determined by an immune cell infiltration assay. Insome embodiments, an anti-CD137 antibody described herein decreasesinfiltration of one or more immune cells into a tumor microenvironmentas determined by an immune cell infiltration assay.

In some embodiments, the immune cell infiltration assay determines aquantity of immune cells expressing one or more immune cell markers in atumor. In some embodiments, the one or more immune cell markers islabeled with an antibody. In some embodiments, the one or more immunecell markers is selected from the group consisting of CD45, CD25, FOXP3,CD4, CD8, F4/80, CD11b, TIGIT and PD-1. In some embodiments, thequantity of immune cells expressing the one or more immune cell markersin a tumor is determined by flow cytometry. Methods of quantifyingimmune cells expressing one or more immune cell markers by flowcytometry are known in the art.

In some embodiments, the anti-CD137 antibody induces or enhancesinfiltration of one or more immune cells into a tumor microenvironmentrelative to a reference antibody, as determined by an immune cellinfiltration assay. In some embodiments, the reference antibody is anantibody comprising the same isotype as the anti-CD137 antibody and doesnot specifically bind to CD137. In some embodiments, the referenceantibody is an antibody comprising the same isotype as the anti-CD137antibody and specifically binds to CD137. In some embodiments, thereference antibody is an antibody comprising the different isotype asthe anti-CD137 antibody and does not specifically bind to CD137. In someembodiments, the reference antibody is an antibody comprising adifferent isotype as the anti-CD137 antibody and specifically binds toCD137.

In some embodiments, an anti-CD137 antibody described herein increasesinfiltration of immune cells expressing CD45 into a tumormicroenvironment in a subject as determined by an immune cellinfiltration assay, wherein the assay comprises the following steps:

(i) administering the anti-CD137 antibody to a subject having a tumor;

(ii) obtaining a sample of the tumor;

(iii) contacting the sample with an fluorescently-labeled detectionantibody that specifically binds to CD45, wherein the detection antibodyfluorescently-labels the immune cells expressing CD45; and

(iv) determining a quantity of the fluorescently-labeled immune cellsexpressing CD45 by flow cytometry,

wherein an increase in the quantity of fluorescently-immune cellsexpressing CD45 in the tumor indicates the anti-CD137 antibody inducesor enhances infiltration of immune cells into the tumormicroenvironment. In some embodiments, an increase in the quantity ofimmune cells expressing CD45 is at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, or 80% of total cells in the tumormicroenvironment.

In some embodiments, an anti-CD137 antibody described herein reduces orinhibits infiltration of one or more immune cells into a tumormicroenvironment as determined by an immune cell infiltration assay. Insome embodiments, the anti-CD137 antibody decreases infiltration of oneor more immune cells into a tumor microenvironment relative to areference antibody, as determined by an immune cell infiltration assay.In some embodiments, the reference antibody is an antibody comprisingthe same isotype as the anti-CD137 antibody and does not specificallybind to CD137. In some embodiments, the reference antibody is anantibody comprising the same isotype as the anti-CD137 antibody andspecifically binds to CD137. In some embodiments, the reference antibodyis an antibody comprising the different isotype as the anti-CD137antibody and does not specifically bind to CD137. In some embodiments,the reference antibody is an antibody comprising a different isotype asthe anti-CD137 antibody and specifically binds to CD137. In someembodiments, a decrease in immune cells is less than 40%, 35%, 30%, 25%,20%, 15%, 10%, or 5% of total cells in a tumor microenvironment.

In some embodiments, an anti-CD137 antibody described herein decreasesinfiltration of tumor associated macrophages into a tumormicroenvironment in a subject as determined by an immune cellinfiltration assay, wherein the assay comprises the following steps:

(i) obtaining a sample of the tumor;

(ii) contacting the sample with one or more antibodies that label thetumor associated macrophage, wherein the one or more antibodiesspecifically bind to an immune cell marker selected from the groupconsisting of F4/80, CD11b, CD45 and a combination thereof; and

(iii) determining a quantity of the labeled tumor associated macrophagesby flow cytometry. In some embodiments, tumor-associated macrophages areless than 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of immune cells inthe tumor microenvironment. In some embodiments, tumor-associatedmacrophages express F4/80, CD11b and CD45.

In some embodiments, an anti-CD137 antibody described herein decreasesinfiltration of T regulatory cells (Tregs) into a tumor microenvironmentin a subject as determined by an immune cell infiltration assay, whereinthe assay comprises the following steps:

(i) obtaining a sample of the tumor;

(ii) contacting the sample with one or more antibodies that label thetumor associated macrophage, wherein the one or more antibodiesspecifically bind to an immune cell marker selected from the groupconsisting of CD25, FOXP-3, CD4 and a combination thereof; and

(iii) determining a quantity of the labeled Treg cells by flowcytometry. In some embodiments, Treg cells are less than 35%, 30%, 25%,20%, 15%, 10%, or 5% of CD4+ T cells in the tumor microenvironment. Insome embodiments, Treg cells express CD4, FOXP-3 and CD25.

In some embodiments, an anti-CD137 antibody described herein protects Tcells from T cell exhaustion and/or reverses T cell exhaustion asdetermined by a T cell exhaustion assay. Exhausted T cells can bedistinguished from other T cell dysfunctions such as anergy andsenescence based on their underlying molecular mechanisms (Crespo etal., (2013) Curr Opin Immunol 25(2):241-221). Whereas anergy occursduring priming due to the absence of costimulatory signals andsenescence is growth arrest after extensive proliferation, exhausted Tcells arise from T cells which initially gained and provided T celleffector function, but that exhibit a gradual deterioration of T celleffector function due to continuous T cell receptor (TCR) stimulationfrom persistent antigen and inflammatory mediators, both of whichcommonly occur in tumors (Wherry & Kurachi (2015) Nat Rev Immunol15(8):486-99). Hallmarks of T cell exhaustion include, but are notlimited to, continuous deterioration of in vivo and/or ex vivo T cellfunction, an increased expression of multiple inhibitory receptors (IRs)(e.g., PD-1, CTLA-4, LAG-3, TIM-3, CD244, CD160, TIGIT), progressiveloss or decrease of effector cytokine secretion (e.g., IL-2, interferongamma (IFNγ), tumor necrosis factor alpha (TNFα)), loss or decrease ofCC chemokine (β-chemokine) production, poor responsiveness to IL-7 andIL-15, loss or decrease of proliferative capacity, loss or decrease ofin vivo and/or ex vivo cytolytic activity, altered cell metabolism and adifferent transcriptional profile relative to non-exhausted T cells.Severely exhausted T cells can succumb to deletion (Yi et al., (2010)Immunology 129(4):474-481).

In some embodiments, an anti-CD137 antibody described herein protects Tcells from T cell exhaustion and/or reverses T cell exhaustion asdetermined by a T cell exhaustion assay wherein the T cell exhaustionassay determines an amount or level of one or more effector cytokinessecreted from T cells treated with an anti-CD137 antibody describedherein, wherein the amount or level of the one or more effectorcytokines indicates protection from or reversion of T cell exhaustion.In some embodiments, the T cell exhaustion assay comprises the followingsteps:

(i) isolating of T cells from a subject (e.g., a human subject);

(ii) contacting the T cells with an antigen that induces T cellexhaustion;

(iii) contacting the T cells with the anti-CD137 antibody;

(iv) determining an amount of one or more effector cytokines secretedfrom the T cells; and;

(v) comparing the amount or level of the one or more effector cytokinessecreted from the T cells to an amount or level secreted from referenceT cells,

wherein the reference T cells are not contacted with the antigen thatinduces T cell exhaustion, and wherein the difference in the amount orlevel of the one or more effector cytokines secreted from the T cellsand reference T cells indicates protection from or reversion of T cellexhaustion.

In some embodiments, the one or more effector cytokines is selected fromIL-2, IFNγ, and TNFα. In some embodiments, the amount or level of theone or more effector cytokines is determined by ELISA. ELISAs suitablefor the determination of the amount or level of the one or more effectorcytokines are known in the art. In some embodiments, the amount or levelof the one or more effector cytokines is determined by Meso ScaleDiscovery. In some embodiments, the amount or level of the one or moreeffector cytokines is determined by any one of the cytokine productionassays described herein.

The gradual dysfunction of exhausted T cells is accompanied by theexpression of IRs, which transmit inhibitory signals to the nucleus uponinteraction with ligands on target cells. Accordingly, in someembodiments, an anti-CD137 antibody described herein protects T cellsfrom T cell exhaustion and/or reverses T cell exhaustion as determinedby a T cell exhaustion assay wherein the T cell exhaustion assaydetermines an expression level of one or more inhibitory receptors on Tcells treated with an anti-CD137 antibody described herein, wherein theexpression level of the one or more inhibitory receptors indicatesprotection from or reversion of T cell exhaustion. In some embodiments,the T cell exhaustion assay comprises the following steps:

(i) isolating of T cells from a subject (e.g., a human subject);

(ii) contacting the T cells with an antigen that induces T cellexhaustion;

(iii) contacting the T cells with the anti-CD137 antibody;

(iv) determining an expression level of one or more inhibitory receptorson T cells; and

(v) comparing the expression level of one or more inhibitory receptorson T cells to an amount or level secreted from reference T cells,wherein the reference T cells are not contacted with the antigen thatinduces T cell exhaustion, and wherein the difference in the expressionlevel of one or more inhibitory receptors on T cells and reference Tcells indicates protection from or reversion of T cell exhaustion.

In some embodiments, the one or more inhibitory receptors is selectedfrom TIGIT and PD-1 In some embodiments, the expression level of the oneor more inhibitory receptors is determined by flow cytometry. Methods todetermine expression levels of inhibitory receptors on immune cells(e.g. T cells) by flow cytometry are known in the art.

In some embodiments, the amount of exhausted T cells is less than 20%,15%, 10% or 5% of total CD8+ or CD4+ T cells in a tumormicroenvironment.

Where the assays described herein refer to ‘isolating T cells from asubject’; it is to be understood that the assay may suitably beperformed on T cells previously isolated from a subject.

Where the assays described herein refer to (i) administering theanti-CD137 antibody to a subject and (ii) isolating T cells from thesubject; it is to be understood that the assay may suitably be performedon T cells previously isolated from a subject to whom the anti-CD137antibody has been administered.

Where the assays described herein refer to ‘obtaining a sample of thetumor’; it is to be understood that the assay may suitably be performedon a sample of a tumor previously isolated from a subject.

Where the assays described herein refer to (i) administering theanti-CD137 antibody to a subject having a tumor and (ii) obtaining asample of the tumor; it is to be understood that the assay may suitablybe performed a sample of a tumor previously isolated from a subject towhom the anti-CD137 antibody has been administered.

III. Non-Ligand Binding

In some embodiments, an anti-CD137 antibody described herein binds to anon-ligand binding region of CD137, as determined by a ligand bindingassay. A ligand binding assay (LBA) is an assay, or an analyticprocedure, that provides a measure of the interactions that occurbetween two reactant molecules (e.g., a receptor and ligandpolypeptides). Suitably, the LBA provides a measure of the degree ofaffinity between the two reactant molecules (e.g., a receptor and ligandpolypeptides). For example, in some embodiments a ligand binding assayis used to determine the presence, rate, extent of binding, orcombinations thereof, of a ligand molecule (e.g., CD137L) to a receptor(e.g., CD137). In some embodiments, to determine the presence, rateand/or extent of ligand binding to a receptor, a ligand binding assaycomprises detecting the formation of a ligand:receptor complex. In someembodiments, to determine the presence, rate and/or extent of ligandbinding to a receptor, a ligand binding assay comprises determining thedissociation of a ligand:receptor complex.

In some embodiments, the formation and/or dissociation of aligand:receptor complex is determined by detection of afluorescently-labeled ligand in complex with a receptor. In someembodiments, the formation and/or dissociation of a ligand:receptorcomplex is determined by detection and/or quantification of an amount offluorescently-labeled receptor in complex with a ligand. In someembodiments, the formation and/or dissociation of a ligand:receptorcomplex is determined by detection and/or quantification of an amount ofa fluorescently-labeled antibody that specifically binds to theligand:receptor complex. Methods of detecting and quantifyingfluorescence are known in the art and include, but are not limited to,fluorescence polarization (FP) and fluorescence anisotropy (FA).

In some embodiments, the formation and/or dissociation of aligand:receptor complex is determined by detection and/or quantificationof an amount of a radioactively-labeled ligand in complex with areceptor. In some embodiments, the formation and/or dissociation of aligand:receptor complex is determined by detection and/or quantificationof an amount of radioactively-labeled receptor in complex with a ligand.In some embodiments, the formation and/or dissociation of aligand:receptor complex is determined by detection and/or quantificationof an amount of a radioactively-labeled antibody that specifically bindsto the ligand:receptor complex. Methods of detecting and quantifyingradioactivity are known in the art and include, but are not limited to,quantitative autoradiography and scintillation counting.

In some embodiments, the formation and/or dissociation of aligand:receptor complex is determined by detection and/or quantificationof an amount of a bioluminescently-labeled ligand in complex with areceptor. In some embodiments, the formation and/or dissociation of aligand:receptor complex is determined by detection and/or quantificationof an amount of bioluminescently-labeled receptor in complex with aligand. In some embodiments, the formation and/or dissociation of aligand:receptor complex is determined by detection and/or quantificationof an amount of a bioluminescently-labeled antibody that specificallybinds to the ligand:receptor complex. Methods of detecting andquantifying bioluminescence are known in the art and include, but arenot limited to, luminometry.

In some embodiment, formation and/or dissociation of the ligand:receptorcomplex is determined by surface plasmon resonance (SPR) as describedsupra.

In some embodiments, a ligand binding assay determines if an antibodythat specifically binds to a receptor (e.g., an anti-CD137 antibody)affects the formation of a ligand:receptor complex by determining thepresence, rate and/or extent of ligand binding to the receptor in thepresence of the antibody. In some embodiments, an antibody (e.g., ananti-CD137 antibody) that specifically binds to a receptor (e.g., CD137)and decreases, disrupts or blocks the formation of a ligand:receptorcomplex (e.g., a CD137:CD137L complex) is known as a “ligand blockingantibody”. In some embodiments, a “ligand blocking antibody” maydecrease the formation of a ligand:receptor complex (e.g., aCD137:CD137L complex) by at least 10%, at least 20%, at least 30%, atleast 40% or at least 50% compared to the formation of theligand:receptor complex (e.g., the CD137:CD137L complex) which occurs inthe absence of the ligand blocking antibody. In some embodiments, anantibody (e.g., an anti-CD137 antibody) that specifically binds to areceptor (e.g., CD137) and does not decrease, disrupt or block theformation of a ligand:receptor complex (e.g., a CD137:CD137L complex) isreferred to as a “non-ligand blocking antibody”. In some embodiments, a“non-ligand blocking antibody” may decrease the formation of aligand:receptor complex (e.g., a CD137:CD137L complex) by less than 10%,less than 5%, less than 2% or less than 1% compared to the formation ofthe ligand:receptor complex (e.g., the CD137:CD137L complex) whichoccurs in the absence of the non-ligand blocking antibody. Accordingly,in some embodiments a ligand binding assay characterizes an antibodythat binds to a receptor as a “ligand blocking antibody” or a“non-ligand blocking antibody”.

In some embodiments, a ligand binding assay characterizes an antibodythat specifically binds to a receptor and promotes the formation of aligand:receptor complex. In some embodiments, a ligand binding assaycharacterizes an antibody that specifically binds to a receptor andstabilizes the formation of a ligand:receptor complex. In someembodiments, the induction and/or stabilization of the formation of aligand:receptor complex by an antibody contributes to the antibody'sagonistic effect. In some embodiments, an anti-CD137 antibody describedherein agonizes CD137, as determined by a ligand binding assay.

In some embodiments, an isolated anti-CD137 antibody, or antigen-bindingfragment thereof, described herein, binds to CD137 and induces CD137Lbinding as determined by a ligand binding assay (LBA).

In some embodiments, an isolated anti-CD137 antibody, or antigen-bindingfragment thereof, described herein, binds to CD137 and induces CD137Lbinding as determined by a ligand binding assay, wherein the ligandbinding assay comprises the following steps:

(i) combining an anti-CD137 antibody with CD137 and CD137L at variousconcentrations, wherein CD137 and CD137L form a CD137:CD137L complex,and

(ii) detecting the CD137:CD137L complex in the presence of theanti-CD137 antibody over time,

wherein an increase in CD137:CD137L complex in the presence of theanti-CD137 antibody indicates the anti-CD137 antibody induces CD137Lbinding to CD137. The increase in CD137:CD137L complex in the presenceof the anti-CD137 antibody may be at least 1.5-fold, at least 2-fold, atleast 5-fold, at least 10-fold, or at least 20-fold greater that theamount CD137:CD137L complex in the absence of the anti-CD137 antibody.

In some embodiments, an isolated anti-CD137 antibody, or antigen-bindingfragment thereof, described herein, binds to a non-ligand binding regionof CD137 as determined by a ligand binding assay, wherein the ligandbinding assay comprises the following steps:

(i) combining an anti-CD137 antibody with CD137 and CD137L at variousconcentrations, wherein CD137 and CD137L form a CD137:CD137L complex,and

(ii) detecting the CD137:CD137L complex in the presence of theanti-CD137 antibody over time,

wherein no change in the CD137:CD137L complex in the presence of theanti-CD137 antibody indicates the anti-CD137 antibody binds to anon-ligand binding region of CD137. In some embodiments, less than a 2%change in CD137:CD137L complex indicates the anti-CD137 antibody bindsto a non-ligand binding region of CD137. In some embodiments, less thana 5% change in CD137:CD137L complex indicates the anti-CD137 antibodybinds to a non-ligand binding region of CD137. In some embodiments, lessthan a 10% change in CD137:CD137L complex indicates the anti-CD137antibody binds to a non-ligand binding region of CD137.

In some embodiments, an anti-CD137 antibody described herein binds to anon-ligand binding region of CD137, as determined by biolayerinterferometry. In some embodiments, an anti-CD137 antibody describedherein binds to a non-ligand binding region of CD137, as determined bysurface plasmon resonance imaging (SPRi). In some embodiments, CD137 andCD137L is sequentially applied to a sensor pre-loaded with an anti-CD137antibody (i.e., the antibody is captured on a sensor). In someembodiments, the binding of an anti-CD137 antibody to a non-ligandbinding region is indicated by an increase in response upon exposure toCD137L.

IV. Functions of CD137 Binding Antibodies

In some embodiments, the anti-CD137 agonist antibodies described hereinbind to human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM) and inhibit or reduce T cellexhaustion. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein bind to human CD137 with an affinity (K_(D)) of about30-100 nM (e.g., between about 30 nM and about 100 nM) and induce orenhance T cell activation. In some embodiments, the anti-CD137 agonistantibodies described herein bind to human CD137 with an affinity (K_(D))of about 30-100 nM (e.g., between about 30 nM and about 100 nM) andinduce or enhance cytokine production by immune cells. In someembodiments, the anti-CD137 agonist antibodies described herein bind tohuman CD137 with an affinity (K_(D)) of about 30-100 nM (e.g., betweenabout 30 nM and about 100 nM) and induce or enhance T cellproliferation. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein bind to human CD137 with an affinity (K_(D)) of about30-100 nM (e.g., between about 30 nM and about 100 nM) and exhibitanti-tumor efficacy. In some embodiments, the anti-CD137 agonistantibodies described herein bind to human CD137 with an affinity (K_(D))of about 30-100 nM (e.g., between about 30 nM and about 100 nM) andinhibit or reduce macrophage differentiation and/or activation. In someembodiments, the anti-CD137 agonist antibodies described herein bind tohuman CD137 with an affinity (K_(D)) of about 30-100 nM (e.g., betweenabout 30 nM and about 100 nM) and induce or enhance NFκβ signaling. Insome embodiments, the anti-CD137 agonist antibodies described hereinbind to human CD137 with an affinity (K_(D)) of about 30-100 nM (e.g.,between about 30 nM and about 100 nM) and induce or enhance immune cellinfiltration into a tumor microenvironment. In some embodiments, theanti-CD137 agonist antibodies described herein bind to human CD137 withan affinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM andabout 100 nM) and do not induce hepatotoxicity. In some embodiments, theanti-CD137 agonist antibodies described herein bind to human CD137 withan affinity (K_(D)) of about 30-100 nM (e.g., between about 30 nM andabout 100 nM) and bind to a non-ligand binding domain on extracellularCD137. In some embodiments, the anti-CD137 agonist antibodies describedherein bind to human CD137 with an affinity (K_(D)) of about 30-100 nM(e.g., between about 30 nM and about 100 nM) and do not inhibit CD137and CD137L interaction. In some embodiments, the anti-CD137 agonistantibodies described herein bind to human CD137 with an affinity (K_(D))of about 30-100 nM (e.g., between about 30 nM and about 100 nM) and bindto an epitope comprising K114 of SEQ ID NO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereininhibit or reduce T cell exhaustion and induce or enhance T cellactivation. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein inhibit or reduce T cell exhaustion and induce orenhance cytokine production by immune cells. In some embodiments, theanti-CD137 agonist antibodies described herein inhibit or reduce T cellexhaustion and induce or enhance T cell proliferation. In someembodiments, the anti-CD137 agonist antibodies described herein inhibitor reduce T cell exhaustion and exhibit anti-tumor efficacy. In someembodiments, the anti-CD137 agonist antibodies described herein inhibitor reduce T cell exhaustion and inhibit or reduce macrophagedifferentiation and/or activation. In some embodiments, the anti-CD137agonist antibodies described herein inhibit or reduce T cell exhaustionand induce or enhance NFκβ signaling. In some embodiments, theanti-CD137 agonist antibodies described herein inhibit or reduce T cellexhaustion and induce or enhance immune cell infiltration into a tumormicroenvironment. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein inhibit or reduce T cell exhaustion and do not inducehepatotoxicity. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein inhibit or reduce T cell exhaustion and bind to anon-ligand binding domain on extracellular CD137. In some embodiments,the anti-CD137 agonist antibodies described herein inhibit or reduce Tcell exhaustion and do not inhibit CD137 and CD137L interaction. In someembodiments, the anti-CD137 agonist antibodies described herein inhibitor reduce T cell exhaustion and bind to an epitope comprising K114 ofSEQ ID NO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereininduce or enhance T cell activation and induce or enhance cytokineproduction by immune cells. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance T cell activation andinduce or enhance T cell proliferation. In some embodiments, theanti-CD137 agonist antibodies described herein induce or enhance T cellactivation and exhibit anti-tumor efficacy. In some embodiments, theanti-CD137 agonist antibodies described herein induce or enhance T cellactivation and inhibit or reduce macrophage differentiation and/oractivation. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein induce or enhance T cell activation and induce orenhance NFκβ signaling. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance T cell activation andinduce or enhance immune cell infiltration into a tumormicroenvironment. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein induce or enhance T cell activation and do not inducehepatotoxicity. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein induce or enhance T cell activation and bind to anon-ligand binding domain on extracellular CD137. In some embodiments,the anti-CD137 agonist antibodies described herein induce or enhance Tcell activation and do not inhibit CD137 and CD137L interaction. In someembodiments, the anti-CD137 agonist antibodies described herein induceor enhance T cell activation and bind to an epitope comprising K114 ofSEQ ID NO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereininduce or enhance cytokine production by immune cells and induce orenhance T cell proliferation. In some embodiments, the anti-CD137agonist antibodies described herein induce or enhance cytokineproduction by immune cells and exhibit anti-tumor efficacy. In someembodiments, the anti-CD137 agonist antibodies described herein induceor enhance cytokine production by immune cells and inhibit or reducemacrophage differentiation and/or activation. In some embodiments, theanti-CD137 agonist antibodies described herein induce or enhancecytokine production by immune cells and induce or enhance NFκβsignaling. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein induce or enhance cytokine production by immune cellsand induce or enhance immune cell infiltration into a tumormicroenvironment. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein induce or enhance cytokine production by immune cellsand do not induce hepatotoxicity. In some embodiments, the anti-CD137agonist antibodies described herein induce or enhance cytokineproduction by immune cells and bind to a non-ligand binding domain onextracellular CD137. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance cytokine production byimmune cells and do not inhibit CD137 and CD137L interaction. In someembodiments, the anti-CD137 agonist antibodies described herein induceor enhance cytokine production by immune cells and bind to an epitopecomprising K114 of SEQ ID NO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereininduce or enhance T cell proliferation and exhibit anti-tumor efficacy.In some embodiments, the anti-CD137 agonist antibodies described hereininduce or enhance T cell proliferation and inhibit or reduce macrophagedifferentiation and/or activation. In some embodiments, the anti-CD137agonist antibodies described herein induce or enhance T cellproliferation and induce or enhance NFκβ signaling. In some embodiments,the anti-CD137 agonist antibodies described herein induce or enhance Tcell proliferation and induce or enhance immune cell infiltration into atumor microenvironment. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance T cell proliferation anddo not induce hepatotoxicity. In some embodiments, the anti-CD137agonist antibodies described herein induce or enhance T cellproliferation and bind to a non-ligand binding domain on extracellularCD137. In some embodiments, the anti-CD137 agonist antibodies describedherein induce or enhance T cell proliferation and do not inhibit CD137and CD137L interaction. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance T cell proliferation andbind to an epitope comprising K114 of SEQ ID NO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereinexhibit anti-tumor efficacy and inhibit or reduce macrophagedifferentiation and/or activation. In some embodiments, the anti-CD137agonist antibodies described herein exhibit anti-tumor efficacy andinduce or enhance NFκβ signaling. In some embodiments, the anti-CD137agonist antibodies described herein exhibit anti-tumor efficacy andinduce or enhance immune cell infiltration into a tumormicroenvironment. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein exhibit anti-tumor efficacy and do not inducehepatotoxicity. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein exhibit anti-tumor efficacy and bind to a non-ligandbinding domain on extracellular CD137. In some embodiments, theanti-CD137 agonist antibodies described herein exhibit anti-tumorefficacy and do not inhibit CD137 and CD137L interaction. In someembodiments, the anti-CD137 agonist antibodies described herein exhibitanti-tumor efficacy and bind to an epitope comprising K114 of SEQ ID NO:3.

In some embodiments, the anti-CD137 agonist antibodies described hereininhibit or reduce macrophage differentiation and/or activation andinduce or enhance NFκβ signaling. In some embodiments, the anti-CD137agonist antibodies described herein inhibit or reduce macrophagedifferentiation and/or activation and induce or enhance immune cellinfiltration into a tumor microenvironment. In some embodiments, theanti-CD137 agonist antibodies described herein inhibit or reducemacrophage differentiation and/or activation and do not inducehepatotoxicity. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein inhibit or reduce macrophage differentiation and/oractivation and bind to a non-ligand binding domain on extracellularCD137. In some embodiments, the anti-CD137 agonist antibodies describedherein inhibit or reduce macrophage differentiation and/or activationand do not inhibit CD137 and CD137L interaction. In some embodiments,the anti-CD137 agonist antibodies described herein inhibit or reducemacrophage differentiation and/or activation and bind to an epitopecomprising K114 of SEQ ID NO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereininduce or enhance NFκβ signaling and induce or enhance immune cellinfiltration into a tumor microenvironment. In some embodiments, theanti-CD137 agonist antibodies described herein induce or enhance NFκβsignaling and do not induce hepatotoxicity. In some embodiments, theanti-CD137 agonist antibodies described herein induce or enhance NFκβsignaling and bind to a non-ligand binding domain on extracellularCD137. In some embodiments, the anti-CD137 agonist antibodies describedherein induce or enhance NFκβ signaling and do not inhibit CD137 andCD137L interaction. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance NFκβ signaling and bind toan epitope comprising K114 of SEQ ID NO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereininduce or enhance immune cell infiltration into a tumor microenvironmentand do not induce hepatotoxicity. In some embodiments, the anti-CD137agonist antibodies described herein induce or enhance immune cellinfiltration into a tumor microenvironment and bind to a non-ligandbinding domain on extracellular CD137. In some embodiments, theanti-CD137 agonist antibodies described herein induce or enhance immunecell infiltration into a tumor microenvironment and do not inhibit CD137and CD137L interaction. In some embodiments, the anti-CD137 agonistantibodies described herein induce or enhance immune cell infiltrationinto a tumor microenvironment and bind to an epitope comprising K114 ofSEQ ID NO: 3. In some embodiments, the anti-CD137 agonist antibodiesdescribed herein do not induce hepatotoxicity and bind to a non-ligandbinding domain on extracellular CD137. In some embodiments, theanti-CD137 agonist antibodies described herein do not inducehepatotoxicity and do not inhibit CD137 and CD137L interaction. In someembodiments, the anti-CD137 agonist antibodies described herein do notinduce hepatotoxicity and bind to an epitope comprising K114 of SEQ IDNO: 3.

In some embodiments, the anti-CD137 agonist antibodies described hereinbind to a non-ligand binding domain on extracellular CD137 and do notinhibit CD137 and CD137L interaction. In some embodiments, theanti-CD137 agonist antibodies described herein bind to a non-ligandbinding domain on extracellular CD137 and bind to an epitope comprisingK114 of SEQ ID NO: 3. In some embodiments, the anti-CD137 agonistantibodies described herein do not inhibit CD137 and CD137L interactionand bind to an epitope comprising K114 of SEQ ID NO: 3.

Epitope Mapping

The disclosure provides anti-CD137 antibodies, or antigen bindingfragments thereof, that specifically bind to an epitope of human CD137and compete with a reference mAb (e.g., mAb1) for binding to the epitopeof human CD137. Methods to characterize, map, or otherwise elucidate theepitope of an anti-CD137 antibody can be grouped into structural,functional, or computational methods. A particularly suitable structuralmethod to determine the precise molecular architecture of theinteraction between an antibody and the corresponding antigen to whichit binds is x-ray crystallography (alternatively “x-rayco-crystallography). A crystal structure of a bonded antibody-antigenpair enables very accurate determination of key interactions betweenindividual amino acids from both side chains and main chain atoms inboth the epitope of the antigen and the paratope of the antibody. Aminoacids that are within 4 angstroms (Å) of each other are generallyconsidered to be contacting residues. The methodology typically involvespurification of antibody and antigen, formation and purification of thecomplex, followed by successive rounds of crystallization screens andoptimization to obtain diffraction-quality crystals. Structural solutionis obtained following x-ray crystallography frequently at a synchrotronsource. Other structural methods for epitope mapping include, but arenot limited to, hydrogen-deuterium exchange coupled to massspectrometry, crosslinking-coupled mass spectrometry, and nuclearmagnetic resonance (NMR) (see, e.g., Epitope Mapping Protocols inMethods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996); Abbottet al., (2014) Immunology 142(4):526-535).

Functional methods for epitope mapping are well known in the art andtypically involve an assessment or quantification of antibody binding towhole proteins, protein fragments or peptides. Functional methods forepitope mapping can be used, for example, to identify linear orconformational epitopes and/or can be used to infer when two or moredistinct antibodies bind to the same or similar epitopes. Functionalmethods for epitope mapping include, for example, immunoblotting andimmunoprecipitation assays, wherein overlapping or contiguous peptidesfrom CD137 are tested for reactivity with an anti-CD137 antibody, e.g.,mAb1. Other functional methods for epitope mapping include array-basedoligopeptide scanning (alternatively known as “overlapping peptidescanning” or “pepscan analysis”), site-directed mutagenesis (e.g.,alanine-scanning mutagenesis), and high-throughput mutagenesis mapping(e.g., shotgun mutagenesis mapping).

Numerous types of competitive binding assays are known, for example:solid phase direct or indirect radioimmunoassay (RIA), solid phasedirect or indirect enzyme immunoassay (EIA), sandwich competition assay(see Stahli et al., Methods in Enzymology 9:242 (1983)); solid phasedirect biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990));and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77(1990)). Typically, such assays involve the use of purified antigenbound to a solid surface or cells and either 1) an unlabeled testantigen-binding protein and a labeled reference antigen-binding protein,or 2) a labeled test antigen-binding protein and an unlabeled referenceantigen-binding protein. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test antigen-binding protein. Usually the testantigen-binding protein is present in excess. Antigen-binding proteinsidentified by competition assay (competing antigen-binding proteins)include antigen-binding proteins binding to the same epitope as thereference antigen-binding proteins (e.g., mAb1) and antigen-bindingproteins binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antigen-binding protein (e.g., mAb1) forsteric hindrance to occur. Additional details regarding methods fordetermining competitive binding are provided in the examples herein.Usually, when a competing antigen-binding protein is present in excess(e.g., about 1-, about 5-, about 10-, about 20-about 50-, or about100-fold excess), it will inhibit (e.g., reduce or block) specificbinding of a reference antigen-binding protein to a common antigen by atleast about 40-45%, about 45-50%, about 50-55%, about 55-60%, about60-65%, about 65-70%, about 70-75% or about 75% or more. In someinstances, binding is inhibited by at least about 80-85%, about 85-90%,about 90-95%, about 95-97%, or about 97% or more.

The site-directed mutagenesis method involves targeted site-directedmutagenesis where critical amino acids are identified by systematicallyintroducing substitutions along the protein sequence and thendetermining the effects of each substitution on antibody binding. Thismay be done by “alanine scanning mutagenesis” (Cunningham and Wells(1989) Science 244:1081-085), or some other form of point mutagenesis ofamino acid residues in CD137. Without being bound by theory, two or moreantibodies (e.g., a test antibody and a reference antibody, e.g., mAb1)have the same epitope if essentially all amino acid mutations in theantigen that reduce or eliminate binding of the first antibody reduce oreliminate binding of the second or more antibodies.

Shotgun mutagenesis mapping utilizes a comprehensive plasmid-mutationlibrary for the target gene, with each clone in the library bearing aunique amino acid mutation and the entire library covering every aminoacid in the target protein. The clones that constitute the mutationlibrary are individually arranged in microplates, expressed withinliving mammalian cells, and tested for immunoreactivity with antibodiesof interest. Amino acids critical for antibody epitopes are identifiedby a loss of reactivity and are then mapped onto a protein structure tovisualize epitopes. Expression of the target protein antigen withinmammalian cells often provides the native structure of the targetprotein antigen, which allows both linear and conformational epitopestructures to be mapped on complex proteins. (Paes et al., J. Am. Chem.Soc. 131 (20): 6952-6954 (2009); Banik and Doranz, Genetic Engineeringand Biotechnology News 3(2): 25-28 (2010)).

The epitope bound by an anti-CD137 antibody may also be determined usingpeptide scanning methods. In peptide scanning, libraries of shortpeptide sequences from overlapping segments of the target protein, CD137are tested for their ability to bind antibodies of interest. Thepeptides are synthesized and screened for binding, e.g. using ELISA orBIACORE, or on a chip, by any of the multiple methods for solid-phasescreening (Reineke et al, Curr. Opin. Biotechnol. 12: 59-64, 2001) as inthe “pepscan” methodology (WO 84/03564; WO 93/09872).

A recently developed technology termed CLIPS (chemical linkage ofpeptides onto scaffolds) may be used to map conformational epitopes. Theloose ends of the peptides are affixed onto synthetic scaffolds, so thatthe scaffolded peptide may be able to adopt the same spatial structureas the corresponding sequence in the intact protein. CLIPS technology isused to fix linear peptides into cyclic structures (‘single-loop’format), and to bring together different parts of a protein binding site(‘double-loop’, ‘triple-loop’, etc. format), so as to createconformational epitopes that may be assayed for antibody binding. (U.S.Pat. No. 7,972,993).

The epitopes bound by antibodies provided by the disclosure may also bemapped using computational methods. In these methods, for example,libraries of peptide fragments are displayed on the surface of the phageor cell. Epitopes are then mapped by screening antibodies against thesefragments using selective binding assays. A number of computationaltools have been developed which allow the prediction of conformationalepitopes based upon linear affinity-selected peptides obtained usingphage display (Mayrose et al., (2007) Bioinformatics 23:3244-3246).Methods are also available for the detection of conformational epitopesby phage display. Microbial display systems may also be used to expressproperly folded antigenic fragments on the cell surface foridentification of conformational epitopes (Cochran et al., J. Immunol.Meth. 287: 147-158, 2004; Rockberg et al., Nature Methods 5: 1039-1045,2008).

Methods involving proteolysis and mass spectroscopy may also be used todetermine antibody epitopes (Baerga-Ortiz et al., Protein Sci. 2002June; 1 1 (6): 1300-1308). In limited proteolysis, the antigen iscleaved by different proteases, in the presence and in the absence ofthe antibody, and the fragments are identified by mass spectrometry. Theepitope is the region of the antigen that becomes protected fromproteolysis upon binding of the antibody (Suckau et al., Proc. Natl.Acad. Sci. USA 87: 9848-9852, 1990). Additional proteolysis basedmethods include, for example, selective chemical modification (Fiedleret al., Bioconjugate Chemistry 1998, 9(2): 236-234, 1998), epitopeexcision (Van de Water et al., Clin. Immunol. Immunopathol. 1997, 85(3):229-235, 1997), and the recently developed method of hydrogen-deuterium(H/D) exchange (Flanagan, N., Genetic Engineering and Biotechnology News3(2): 25-28, 2010).

In some embodiments, the anti-CD137 antibodies described herein bind toan epitope located within amino acid residues 111-135 of SEQ ID NO: 3 asdetermined by mutagenesis and mammalian display. In some embodiments,the anti-CD137 antibodies described herein bind to an epitope comprisingK114 of SEQ ID NO: 3 as determined by mutagenesis and mammalian display.In some embodiments, the anti-CD137 antibodies described herein bind toan epitope comprising E111, T113 and K114 of SEQ ID NO: 3 as determinedby mutagenesis and mammalian display. In some embodiments, theanti-CD137 antibodies described herein bind to an epitope comprisingE111, T113, K114 and P135 of SEQ ID NO: 3 as determined by mutagenesisand mammalian display. In some embodiments, the anti-CD137 antibodiesdescribed herein bind to an epitope comprising E111, T113, K114, N126,I132 and P135 of SEQ ID NO: 3 as determined by mutagenesis and mammaliandisplay.

Methods for Producing the Anti-CD137 Antibodies and Antigen-BindingFragments Thereof

The disclosure also features methods for producing any of the anti-CD137antibodies or antigen-binding fragments thereof described herein. Insome embodiments, methods for preparing an antibody described herein caninclude immunizing a subject (e.g., a non-human mammal) with anappropriate immunogen. Suitable immunogens for generating any of theantibodies described herein are set forth herein. For example, togenerate an antibody that binds to CD137, a skilled artisan can immunizea suitable subject (e.g., a non-human mammal such as a rat, a mouse, agerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-humanprimate) with a full-length CD137 polypeptide such as a full-lengthhuman CD137 polypeptide comprising the amino acid sequence depicted inSEQ ID NO. 3.

A suitable subject (e.g., a non-human mammal) can be immunized with theappropriate antigen along with subsequent booster immunizations a numberof times sufficient to elicit the production of an antibody by themammal. The immunogen can be administered to a subject (e.g., anon-human mammal) with an adjuvant. Adjuvants useful in producing anantibody in a subject include, but are not limited to, proteinadjuvants; bacterial adjuvants, e.g., whole bacteria (BCG,Corynebacterium parvum or Salmonella minnesota) and bacterial componentsincluding cell wall skeleton, trehalose dimycolate, monophosphoryl lipidA, methanol extractable residue (MER) of tubercle bacillus, complete orincomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g.,aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Otheradjuvants that can be used in the methods for inducing an immuneresponse include, e.g., cholera toxin and parapoxvirus proteins. Seealso Bieg et al. (1999) Autoimmunity 31(1):15-24. See also, e.g.,Lodmell et al. (2000) Vaccine 18:1059-1066; Johnson et al. (1999) J MedChem 42:4640-4649; Baldridge et al. (1999) Methods 19:103-107; and Guptaet al. (1995) Vaccine 13(14): 1263-1276.

In some embodiments, the methods include preparing a hybridoma cell linethat secretes a monoclonal antibody that binds to the immunogen. Forexample, a suitable mammal such as a laboratory mouse is immunized witha CD137 polypeptide as described above. Antibody-producing cells (e.g.,B cells of the spleen) of the immunized mammal can be isolated two tofour days after at least one booster immunization of the immunogen andthen grown briefly in culture before fusion with cells of a suitablemyeloma cell line. The cells can be fused in the presence of a fusionpromoter such as, e.g., vaccinia virus or polyethylene glycol. Thehybrid cells obtained in the fusion are cloned, and cell clonessecreting the desired antibodies are selected. For example, spleen cellsof Balb/c mice immunized with a suitable immunogen can be fused withcells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14.After the fusion, the cells are expanded in suitable culture medium,which is supplemented with a selection medium, for example HAT medium,at regular intervals in order to prevent normal myeloma cells fromovergrowing the desired hybridoma cells. The obtained hybridoma cellsare then screened for secretion of the desired antibodies, e.g., anantibody that binds to CD137.

In some embodiments, a skilled artisan can identify an anti-CD137antibody from a non-immune biased library as described in, e.g., U.S.Pat. No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt etal. (2005) Nucleic Acids Res 33(9):e81.

In some embodiments, the methods described herein can involve, or beused in conjunction with, e.g., phage display technologies, bacterialdisplay, yeast surface display, eukaryotic viral display, mammalian celldisplay, and cell-free (e.g., ribosomal display) antibody screeningtechniques (see, e.g., Etz et al. (2001) J Bacteriol 183:6924-6935;Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000)Microbiology 146:3025-3032; Kieke et al. (1997) Protein Eng10:1303-1310; Yeung et al. (2002) Biotechnol Prog 18:212-220; Boder etal. (2000) Methods Enzymology 328:430-444; Grabherr et al. (2001) CombChem High Throughput Screen 4:185-192; Michael et al. (1995) Gene Ther2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; Schaffitzel etal. (1999) J Immunol Methods 231:119-135; and Hanes et al. (2000) NatBiotechnol 18:1287-1292).

Methods for identifying antibodies using various phage display methodsare known in the art. In phage display methods, functional antibodydomains are displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. Such phage can be utilized todisplay antigen-binding domains of antibodies, such as Fab, Fv, ordisulfide-bond stabilized Fv antibody fragments, expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Phage used in these methods are typically filamentous phage such as fdand M13. The antigen binding domains are expressed as arecombinantly-fused protein to any of the phage coat proteins pIII,pVIII, or pIX. See, e.g., Shi et al. (2010) JMB 397:385-396. Examples ofphage display methods that can be used to make the immunoglobulins, orfragments thereof, described herein include those disclosed in Brinkmanet al. (1995) J Immunol Methods 182:41-50; Ames et al. (1995) J ImmunolMethods 184:177-186; Kettleborough et al. (1994) Eur J Immunol24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al. (1994)Advances in Immunology 57:191-280; and PCT publication nos. WO 90/02809,WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO95/20401. Suitable methods are also described in, e.g., U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108.

In some embodiments, the phage display antibody libraries can begenerated using mRNA collected from B cells from the immunized mammals.For example, a splenic cell sample comprising B cells can be isolatedfrom mice immunized with CD137 polypeptide as described above. mRNA canbe isolated from the cells and converted to cDNA using standardmolecular biology techniques. See, e.g., Sambrook et al. (1989)“Molecular Cloning: A Laboratory Manual, 2^(nd) Edition,” Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane(1988), supra; Benny K. C. Lo (2004), supra; and Borrebaek (1995),supra. The cDNA coding for the variable regions of the heavy chain andlight chain polypeptides of immunoglobulins are used to construct thephage display library. Methods for generating such a library aredescribed in, e.g., Merz et al. (1995) J Neurosci Methods 62(1-2):213-9;Di Niro et al. (2005) Biochem J 388(Pt L:889-894; and Engberg et al.(1995) Methods Mol Biol 51:355-376.

In some embodiments, a combination of selection and screening can beemployed to identify an antibody of interest from, e.g., a population ofhybridoma-derived antibodies or a phage display antibody library.Suitable methods are known in the art and are described in, e.g.,Hoogenboom (1997) Trends in Biotechnology 15:62-70; Brinkman et al.(1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994),supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. Forexample, a plurality of phagemid vectors, each encoding a fusion proteinof a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage)and a different antigen-combining region are produced using standardmolecular biology techniques and then introduced into a population ofbacteria (e.g., E. coli). Expression of the bacteriophage in bacteriacan, in some embodiments, require use of a helper phage. In someembodiments, no helper phage is required (see, e.g., Chasteen et al.,(2006) Nucleic Acids Res 34(21):e145). Phage produced from the bacteriaare recovered and then contacted to, e.g., a target antigen bound to asolid support (immobilized). Phage may also be contacted to antigen insolution, and the complex is subsequently bound to a solid support.

A subpopulation of antibodies screened using the above methods can becharacterized for their specificity and binding affinity for aparticular antigen (e.g., human CD137) using any immunological orbiochemical based method known in the art. For example, specific bindingof an antibody to CD137, may be determined for example usingimmunological or biochemical based methods such as, but not limited to,an ELISA assay, SPR assays, immunoprecipitation assay, affinitychromatography, and equilibrium dialysis as described above.Immunoassays which can be used to analyze immunospecific binding andcross-reactivity of the antibodies include, but are not limited to,competitive and non-competitive assay systems using techniques such asWestern blots, RIA, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, immunodiffusionassays, agglutination assays, complement-fixation assays,immunoradiometric assays, fluorescent immunoassays, and protein Aimmunoassays. Such assays are routine and well known in the art.

It is understood that the above methods can also be used to determineif, e.g., an anti-CD137 antibody does not bind to full-length, humanCD137 and/or CD137 proteins.

In embodiments where the selected CDR amino acid sequences are shortsequences (e.g., fewer than 10-15 amino acids in length), nucleic acidsencoding the CDRs can be chemically synthesized as described in, e.g.,Shiraishi et al. (2007) Nucleic Acids Symposium Series 51(1):129-130 andU.S. Pat. No. 6,995,259. For a given nucleic acid sequence encoding anacceptor antibody, the region of the nucleic acid sequence encoding theCDRs can be replaced with the chemically synthesized nucleic acids usingstandard molecular biology techniques. The 5′ and 3′ ends of thechemically synthesized nucleic acids can be synthesized to comprisesticky end restriction enzyme sites for use in cloning the nucleic acidsinto the nucleic acid encoding the variable region of the donorantibody. Alternatively, fragments of chemically synthesized nucleicacids, together capable of encoding an antibody, can be joined togetherusing DNA assembly techniques known in the art (e.g. Gibson Assembly).

In some embodiments, the anti-CD137 antibodies described herein comprisean altered heavy chain constant region that has reduced (or no) effectorfunction relative to its corresponding unaltered constant region.Effector functions involving the constant region of the anti-CD137antibody may be modulated by altering properties of the constant or Fcregion. Altered effector functions include, for example, a modulation inone or more of the following activities: antibody-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis,binding to one or more Fc-receptors, and pro-inflammatory responses.Modulation refers to an increase, decrease, or elimination of aneffector function activity exhibited by a subject antibody containing analtered constant region as compared to the activity of the unalteredform of the constant region. In particular embodiments, modulationincludes situations in which an activity is abolished or completelyabsent.

An altered constant region with altered FcR binding affinity and/or ADCCactivity and/or altered CDC activity is a polypeptide which has eitheran enhanced or diminished FcR binding activity and/or ADCC activityand/or CDC activity compared to the unaltered form of the constantregion. An altered constant region which displays increased binding toan FcR binds at least one FcR with greater affinity than the unalteredpolypeptide. An altered constant region which displays decreased bindingto an FcR binds at least one FcR with lower affinity than the unalteredform of the constant region. Such variants which display decreasedbinding to an FcR may possess little or no appreciable binding to anFcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42,41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1%) of the binding to the FcR as compared to the level ofbinding of a native sequence immunoglobulin constant or Fc region to theFcR. Similarly, an altered constant region that displays modulated ADCCand/or CDC activity may exhibit either increased or reduced ADCC and/orCDC activity compared to the unaltered constant region. For example, insome embodiments, the anti-CD137 antibody comprising an altered constantregion can exhibit approximately 0 to 50% (e.g., less than 50, 49, 48,47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30,29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the ADCC and/or CDC activityof the unaltered form of the constant region. An anti-CD137 antibodydescribed herein comprising an altered constant region displayingreduced ADCC and/or CDC may exhibit reduced or no ADCC and/or CDCactivity.

In some embodiments, an anti-CD137 antibody described herein exhibitsreduced or no effector function. In some embodiments, an anti-CD137antibody comprises a hybrid constant region, or a portion thereof, suchas a G2/G4 hybrid constant region (see e.g., Burton et al. (1992) AdvImmun 51:1-18; Canfield et al. (1991) J Exp Med 173:1483-1491; andMueller et al. (1997) Mol Immunol 34(6):441-452). See above.

In some embodiments, an anti-CD137 antibody may contain an alteredconstant region exhibiting enhanced or reduced complement dependentcytotoxicity (CDC). Modulated CDC activity may be achieved byintroducing one or more amino acid substitutions, insertions, ordeletions in an Fc region of the antibody. See, e.g., U.S. Pat. No.6,194,551. Alternatively or additionally, cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved or reduced internalization capability and/or increased ordecreased complement-mediated cell killing. See, e.g., Caron et al.(1992) J Exp Med 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922;PCT publication nos. WO 99/51642 and WO 94/29351; Duncan and Winter(1988) Nature 322:738-40; and U.S. Pat. Nos. 5,648,260 and 5,624,821.

Recombinant Antibody Expression and Purification

The antibodies or antigen-binding fragments thereof described herein canbe produced using a variety of techniques known in the art of molecularbiology and protein chemistry. For example, a nucleic acid encoding oneor both of the heavy and light chain polypeptides of an antibody can beinserted into an expression vector that contains transcriptional andtranslational regulatory sequences, which include, e.g., promotersequences, ribosomal binding sites, transcriptional start and stopsequences, translational start and stop sequences, transcriptionterminator signals, polyadenylation signals, and enhancer or activatorsequences. The regulatory sequences include a promoter andtranscriptional start and stop sequences. In addition, the expressionvector can include more than one replication system such that it can bemaintained in two different organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification.

Several possible vector systems are available for the expression ofcloned heavy chain and light chain polypeptides from nucleic acids inmammalian cells. One class of vectors relies upon the integration of thedesired gene sequences into the host cell genome. Cells which havestably integrated DNA can be selected by simultaneously introducing drugresistance genes such as E. coli gpt (Mulligan and Berg (1981) Proc NatlAcad Sci USA 78:2072) or Tn5 neo (Southern and Berg (1982) Mol ApplGenet 1:327). The selectable marker gene can be either linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection (Wigler et al. (1979) Cell 16:77). A second class ofvectors utilizes DNA elements which confer autonomously replicatingcapabilities to an extrachromosomal plasmid. These vectors can bederived from animal viruses, such as bovine papillomavirus (Sarver etal. (1982) Proc Natl Acad Sci USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (1984) Proc Natl Acad Sci USA 81:1292), or SV40virus (Lusky and Botchan (1981) Nature 293:79).

The expression vectors can be introduced into cells in a manner suitablefor subsequent expression of the nucleic acid. The method ofintroduction is largely dictated by the targeted cell type, discussedbelow. Exemplary methods include CaPO4 precipitation, liposome fusion,cationic liposomes, electroporation, viral infection, dextran-mediatedtransfection, polybrene-mediated transfection, protoplast fusion, anddirect microinjection.

Appropriate host cells for the expression of antibodies orantigen-binding fragments thereof include yeast, bacteria, insect,plant, and mammalian cells. Of particular interest are bacteria such asE. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris,insect cells such as SF9, mammalian cell lines (e.g., human cell lines),as well as primary cell lines.

In some embodiments, an antibody or fragment thereof can be expressedin, and purified from, transgenic animals (e.g., transgenic mammals).For example, an antibody can be produced in transgenic non-human mammals(e.g., rodents) and isolated from milk as described in, e.g., Houdebine(2002) Curr Opin Biotechnol 13(6):625-629; van Kuik-Romeijn et al.(2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J ImmunolMethods 231(1-2):147-157.

The antibodies and fragments thereof can be produced from the cells byculturing a host cell transformed with the expression vector containingnucleic acid encoding the antibodies or fragments, under conditions, andfor an amount of time, sufficient to allow expression of the proteins.Such conditions for protein expression will vary with the choice of theexpression vector and the host cell, and will be easily ascertained byone skilled in the art through routine experimentation. For example,antibodies expressed in E. coli can be refolded from inclusion bodies(see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expressionsystems and methods for their use are well known in the art (see CurrentProtocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—ALaboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York(2001)). The choice of codons, suitable expression vectors and suitablehost cells will vary depending on a number of factors, and may be easilyoptimized as needed. An antibody (or fragment thereof) described hereincan be expressed in mammalian cells or in other expression systemsincluding but not limited to yeast, baculovirus, and in vitro expressionsystems (see, e.g., Kaszubska et al. (2000) Protein Expression andPurification 18:213-220).

Following expression, the antibodies and fragments thereof can beisolated. An antibody or fragment thereof can be isolated or purified ina variety of ways known to those skilled in the art depending on whatother components are present in the sample. Standard purificationmethods include electrophoretic, molecular, immunological, andchromatographic techniques, including ion exchange, hydrophobic,affinity, and reverse-phase HPLC chromatography. For example, anantibody can be purified using a standard anti-antibody column (e.g., aprotein-A or protein-G column). Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.See, e.g., Scopes (1994) “Protein Purification, 3^(rd) edition,”Springer-Verlag, New York City, N.Y. The degree of purificationnecessary will vary depending on the desired use. In some instances, nopurification of the expressed antibody or fragments thereof will benecessary.

Methods for determining the yield or purity of a purified antibody orfragment thereof are known in the art and include, e.g., Bradford assay,UV spectroscopy, Biuret protein assay, Lowry protein assay, amido blackprotein assay, high pressure liquid chromatography (HPLC), massspectrometry (MS), and gel electrophoretic methods (e.g., using aprotein stain such as Coomassie Blue or colloidal silver stain).

Modification of the Antibodies or Antigen-Binding Fragments Thereof

The antibodies or antigen-binding fragments thereof can be modifiedfollowing their expression and purification. The modifications can becovalent or non-covalent modifications. Such modifications can beintroduced into the antibodies or fragments by, e.g., reacting targetedamino acid residues of the polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues. Suitable sites for modification can be chosen using any of avariety of criteria including, e.g., structural analysis or amino acidsequence analysis of the antibodies or fragments.

In some embodiments, the antibodies or antigen-binding fragments thereofcan be conjugated to a heterologous moiety. The heterologous moiety canbe, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxinor a drug), or a detectable label such as, but not limited to, aradioactive label, an enzymatic label, a fluorescent label, a heavymetal label, a luminescent label, or an affinity tag such as biotin orstreptavidin. Suitable heterologous polypeptides include, e.g., anantigenic tag (e.g., FLAG (DYKDDDDK; SEQ ID NO: 98), polyhistidine(6-His; HHHHHH; SEQ ID NO: 99), hemagglutinin (HA; YPYDVPDYA; SEQ ID NO:100), glutathione-S-transferase (GST), or maltose-binding protein (MBP))for use in purifying the antibodies or fragments. Heterologouspolypeptides also include polypeptides (e.g., enzymes) that are usefulas diagnostic or detectable markers, for example, luciferase, afluorescent protein (e.g., green fluorescent protein (GFP)), orchloramphenicol acetyl transferase (CAT). Suitable radioactive labelsinclude, ³²P, ³³P, ¹⁴C, ¹²⁵I, ¹³¹I, ³⁵S, and ³H. Suitable fluorescentlabels include, without limitation, fluorescein, fluoresceinisothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488,phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700,Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any ofa variety of luminescent lanthanide (e.g., europium or terbium)chelates. For example, suitable europium chelates include the europiumchelate of diethylene triamine pentaacetic acid (DTPA) ortetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labelsinclude, e.g., alkaline phosphatase, CAT, luciferase, and horseradishperoxidase.

Two proteins (e.g., an antibody and a heterologous moiety) can becross-linked using any of a number of known chemical cross linkers.Examples of such cross linkers are those which link two amino acidresidues via a linkage that includes a “hindered” disulfide bond. Inthese linkages, a disulfide bond within the cross-linking unit isprotected (by hindering groups on either side of the disulfide bond)from reduction by the action, for example, of reduced glutathione or theenzyme disulfide reductase. One suitable reagent,4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio) toluene (SMPT),forms such a linkage between two proteins utilizing a terminal lysine onone of the proteins and a terminal cysteine on the other.Heterobifunctional reagents that cross-link by a different couplingmoiety on each protein can also be used. Other useful cross-linkersinclude, without limitation, reagents which link two amino groups (e.g.,N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g.,1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and acarboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an aminogroup and a guanidinium group that is present in the side chain ofarginine (e.g., p-azidophenyl glyoxal monohydrate).

In some embodiments, a radioactive label can be directly conjugated tothe amino acid backbone of the antibody. Alternatively, the radioactivelabel can be included as part of a larger molecule (e.g., ¹²⁵I inmeta-[¹²⁵]iodophenyl-N-hydroxysuccinimide ([¹²⁵I]mIPNHS) which binds tofree amino groups to form meta-iodophenyl (mIP) derivatives of relevantproteins (see, e.g., Rogers et al. (1997) J Nucl Med 38:1221-1229) orchelate (e.g., to DOTA or DTPA) which is in turn bound to the proteinbackbone. Methods of conjugating the radioactive labels or largermolecules/chelates containing them to the antibodies or antigen-bindingfragments described herein are known in the art. Such methods involveincubating the proteins with the radioactive label under conditions(e.g., pH, salt concentration, and/or temperature) that facilitatebinding of the radioactive label or chelate to the protein (see, e.g.,U.S. Pat. No. 6,001,329).

Methods for conjugating a fluorescent label (sometimes referred to as a“fluorophore”) to a protein (e.g., an antibody) are known in the art ofprotein chemistry. For example, fluorophores can be conjugated to freeamino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines)of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP)ester moieties attached to the fluorophores. In some embodiments, thefluorophores can be conjugated to a heterobifunctional cross-linkermoiety such as sulfo-SMCC. Suitable conjugation methods involveincubating an antibody protein, or fragment thereof, with thefluorophore under conditions that facilitate binding of the fluorophoreto the protein. See, e.g., Welch and Redvanly (2003) “Handbook ofRadiopharmaceuticals: Radiochemistry and Applications,” John Wiley andSons (ISBN 0471495603).

In some embodiments, the antibodies or fragments can be modified, e.g.,with a moiety that improves the stabilization and/or retention of theantibodies in circulation, e.g., in blood, serum, or other tissues. Forexample, the antibody or fragment can be PEGylated as described in,e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al.(2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al.(2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (FreseniusKabi, Germany; see, e.g., Pavisie et al. (2010) Int J Pharm387(1-2):110-119). The stabilization moiety can improve the stability,or retention of, the antibody (or fragment) by at least 1.5 (e.g., atleast 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.

In some embodiments, the antibodies or antigen-binding fragments thereofdescribed herein can be glycosylated. In some embodiments, an antibodyor antigen-binding fragment thereof described herein can be subjected toenzymatic or chemical treatment, or produced from a cell, such that theantibody or fragment has reduced or absent glycosylation. Methods forproducing antibodies with reduced glycosylation are known in the art anddescribed in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991) EMBO J10(10):2717-2723; and Co et al. (1993) Mol Immunol 30:1361.

Pharmaceutical Compositions and Formulations

In certain embodiments, the invention provides for a pharmaceuticalcomposition comprising an anti-CD137 antibody with a pharmaceuticallyacceptable diluent, carrier, solubilizer, emulsifier, preservativeand/or adjuvant.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the formulation material(s) are for s.c. and/orI.V. administration. In certain embodiments, the pharmaceuticalcomposition can contain formulation materials for modifying, maintainingor preserving, for example, the pH, osmolality, viscosity, clarity,color, isotonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption or penetration of the composition. In certainembodiments, suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine orlysine); antimicrobials; antioxidants (such as ascorbic acid, sodiumsulfite or sodium hydrogen-sulfite); buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates or other organic acids);bulking agents (such as mannitol or glycine); chelating agents (such asethylenediamine tetraacetic acid (EDTA)); complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company (1995). In certain embodiments, the formulationcomprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH5.2, 9% Sucrose. In certain embodiments, the optimal pharmaceuticalcomposition will be determined by one skilled in the art depending upon,for example, the intended route of administration, delivery format anddesired dosage. See, for example, Remington's Pharmaceutical Sciences,supra. In certain embodiments, such compositions may influence thephysical state, stability, rate of in vivo release and/or rate of invivo clearance of the anti-CD137 antibody.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Incertain embodiments, the saline comprises isotonic phosphate-bufferedsaline. In certain embodiments, neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In certainembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can furtherinclude sorbitol or a suitable substitute therefore. In certainembodiments, a composition comprising an anti-CD137 antibody can beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, a compositioncomprising an anti-CD137 antibody can be formulated as a lyophilizateusing appropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising an anti-CD137antibody, in a pharmaceutically acceptable vehicle. In certainembodiments, a vehicle for parenteral injection is sterile distilledwater in which an anti-CD137 antibody is formulated as a sterile,isotonic solution, and properly preserved. In certain embodiments, thepreparation can involve the formulation of the desired molecule with anagent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that can provide for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. In certain embodiments, hyaluronic acid can also be used, andcan have the effect of promoting sustained duration in the circulation.In certain embodiments, implantable drug delivery devices can be used tointroduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, an anti-CD137 antibody can beformulated as a dry powder for inhalation. In certain embodiments, aninhalation solution comprising an anti-CD137 antibody can be formulatedwith a propellant for aerosol delivery. In certain embodiments,solutions can be nebulized. Pulmonary administration is furtherdescribed in PCT application No. PCT/US94/001875, which describespulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, an anti-CD137 antibody thatis administered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In certain embodiments, at leastone additional agent can be included to facilitate absorption of ananti-CD137 antibody. In certain embodiments, diluents, flavorings, lowmelting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of an anti-CD137 antibody in a mixture with non-toxicexcipients which are suitable for the manufacture of tablets. In certainembodiments, by dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions can be prepared in unit-dose form. Incertain embodiments, suitable excipients include, but are not limitedto, inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving an anti-CD137 antibody insustained- or controlled-delivery formulations. In certain embodiments,techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising an anti-CD137 antibody to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which an anti-CD137 antibody is beingused, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinician can titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of an anti-CD137 antibody in theformulation used. In certain embodiments, a clinician will administerthe composition until a dosage is reached that achieves the desiredeffect. In certain embodiments, the composition can therefore beadministered as a single dose or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. In certain embodiments, appropriate dosages can beascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.In certain embodiments, individual elements of the combination therapymay be administered by different routes.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration. In certain embodiments, it can be desirable to use apharmaceutical composition comprising an anti-CD137 antibody in an exvivo manner. In such instances, cells, tissues and/or organs that havebeen removed from the patient are exposed to a pharmaceuticalcomposition comprising an anti-CD137 antibody after which the cells,tissues and/or organs are subsequently implanted back into the patient.

In certain embodiments, an anti-CD137 antibody can be delivered byimplanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptides. In certain embodiments, such cells can be animal or humancells, and can be autologous, heterologous, or xenogeneic. In certainembodiments, the cells can be immortalized. In certain embodiments, inorder to decrease the chance of an immunological response, the cells canbe encapsulated to avoid infiltration of surrounding tissues. In certainembodiments, the encapsulation materials are typically biocompatible,semi-permeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

Applications

The compositions described herein can be used in diagnostic andtherapeutic applications. For example, detectably-labeledantigen-binding molecules can be used in assays to detect the presenceor amount of the target antigens in a sample (e.g., a biologicalsample). The compositions can be used in in vitro assays for studyinginhibition of target antigen function (e.g. CD137-mediated cellularsignaling or response). In some embodiments, e.g., in which thecompositions bind to and activate a target antigen (e.g. a protein orpolypeptide), the compositions can be used as positive controls inassays designed to identify additional novel compounds that also induceactivity of the target protein or polypeptide and/or are otherwise areuseful for treating a disorder associated with the target protein orpolypeptide. For example, a CD137-activating composition can be used asa positive control in an assay to identify additional compounds (e.g.,small molecules, aptamers, or antibodies) that induce, increase, orstimulate CD137 function. The compositions can also be used intherapeutic methods as elaborated on below.

Kits

In some embodiments, the disclosure provides a kit comprising ananti-CD137 antibody described herein. In some embodiments, a kitincludes an anti-CD137 antibody as disclosed herein, and instructionsfor use. The kits may comprise, in a suitable container, an anti-CD137antibody, one or more controls, and various buffers, reagents, enzymesand other standard ingredients well known in the art.

The container can include at least one vial, well, test tube, flask,bottle, syringe, or other container means, into which an anti-CD137antibody may be placed, and in some instances, suitably aliquoted. Wherean additional component is provided, the kit can contain additionalcontainers into which this component may be placed. The kits can alsoinclude a means for containing an anti-CD137 antibody and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained. Containers and/or kits can includelabeling with instructions for use and/or warnings.

In some embodiments, a kit comprises a containing comprising ananti-CD137 antibody and a pharmaceutically acceptable carrier, or apharmaceutical composition comprising the anti-CD137 antibody, andinstructions for treating or delaying progression of cancer or reducingor inhibiting tumor growth in a subject in need thereof. In someembodiments, a kit comprises a containing comprising an anti-CD137antibody and a pharmaceutically acceptable carrier, or a pharmaceuticalcomposition comprising the anti-CD137 antibody, and instructions foradministering the anti-CD137 antibody to a subject in need thereof,alone or in combination with another agent, for treating or delayingprogression of cancer or reducing or inhibiting tumor growth in thesubject.

Methods of Use

The compositions of the present invention have numerous in vitro and invivo utilities involving the detection and/or quantification of CD137and/or the agonism of CD137 function.

The above-described compositions are useful in, inter alia, methods fortreating or preventing a variety of cancers in a subject. Thecompositions can be administered to a subject, e.g., a human subject,using a variety of methods that depend, in part, on the route ofadministration. The route can be, e.g., intravenous injection orinfusion (IV), subcutaneous injection (SC), intraperitoneal (IP)injection, intramuscular injection (IM), or intrathecal injection (IT).The injection can be in a bolus or a continuous infusion.

Administration can be achieved by, e.g., local infusion, injection, orby means of an implant. The implant can be of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. The implant can be configured for sustained or periodicrelease of the composition to the subject. See, e.g., U.S. PatentApplication Publication No. 20080241223; U.S. Pat. Nos. 5,501,856;4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures ofeach of which are incorporated herein by reference in their entirety.The composition can be delivered to the subject by way of an implantabledevice based on, e.g., diffusive, erodible, or convective systems, e.g.,osmotic pumps, biodegradable implants, electrodiffusion systems,electroosmosis systems, vapor pressure pumps, electrolytic pumps,effervescent pumps, piezoelectric pumps, erosion-based systems, orelectromechanical systems.

In some embodiments, an anti-CD137 antibody or antigen-binding fragmentthereof is therapeutically delivered to a subject by way of localadministration.

A suitable dose of an antibody or fragment thereof described herein,which dose is capable of treating or preventing cancer in a subject, candepend on a variety of factors including, e.g., the age, sex, and weightof a subject to be treated and the particular inhibitor compound used.For example, a different dose of a whole anti-CD137 antibody may berequired to treat a subject with cancer as compared to the dose of aCD137-binding Fab′ antibody fragment required to treat the same subject.Other factors affecting the dose administered to the subject include,e.g., the type or severity of the cancer. For example, a subject havingmetastatic melanoma may require administration of a different dosage ofan anti-CD137 antibody than a subject with glioblastoma. Other factorscan include, e.g., other medical disorders concurrently or previouslyaffecting the subject, the general health of the subject, the geneticdisposition of the subject, diet, time of administration, rate ofexcretion, drug combination, and any other additional therapeutics thatare administered to the subject. It should also be understood that aspecific dosage and treatment regimen for any particular subject willalso depend upon the judgment of the treating medical practitioner(e.g., doctor or nurse). Suitable dosages are described herein. In someembodiments, the anti-CD137 antibodies described herein are effective atboth high and low doses.

A pharmaceutical composition can include a therapeutically effectiveamount of an anti-CD137 antibody or antigen-binding fragment thereofdescribed herein. Such effective amounts can be readily determined byone of ordinary skill in the art based, in part, on the effect of theadministered antibody, or the combinatorial effect of the antibody andone or more additional active agents, if more than one agent is used. Atherapeutically effective amount of an antibody or fragment thereofdescribed herein can also vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody (and one or more additional active agents) to elicit a desiredresponse in the individual, e.g., reduction in tumor growth. Forexample, a therapeutically effective amount of an anti-CD137 antibodycan inhibit (lessen the severity of or eliminate the occurrence of)and/or prevent a particular disorder, and/or any one of the symptoms ofthe particular disorder known in the art or described herein. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the composition are outweighed by thetherapeutically beneficial effects.

Suitable human doses of any of the antibodies or fragments thereofdescribed herein can further be evaluated in, e.g., Phase I doseescalation studies. See, e.g., van Gurp et al. (2008) Am JTransplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res13(2, part 1):523-531; and Hetherington et al. (2006) AntimicrobialAgents and Chemotherapy 50(10): 3499-3500.

In some embodiments, the composition contains any of the antibodies orantigen-binding fragments thereof described herein and one or more(e.g., two, three, four, five, six, seven, eight, nine, 10, or 11 ormore) additional therapeutic agents such that the composition as a wholeis therapeutically effective. For example, a composition can contain ananti-CD137 antibody described herein and an alkylating agent, whereinthe antibody and agent are each at a concentration that when combinedare therapeutically effective for treating or preventing a cancer (e.g.,melanoma) in a subject.

Toxicity and therapeutic efficacy of such compositions can be determinedby known pharmaceutical procedures in cell cultures or experimentalanimals (e.g., animal models of any of the cancers described herein).These procedures can be used, e.g., for determining the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. An antibody or antigen-binding fragment thereofthat exhibits a high therapeutic index is preferred. While compositionsthat exhibit toxic side effects may be used, care should be taken todesign a delivery system that targets such compounds to the site ofaffected tissue and to minimize potential damage to normal cells and,thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch antibodies or antigen-binding fragments thereof lies generallywithin a range of circulating concentrations of the antibodies orfragments that include the ED₅₀ with little or no toxicity. The dosagemay vary within this range depending upon the dosage form employed andthe route of administration utilized. For an anti-CD137 antibodydescribed herein, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe EC₅₀ (i.e., the concentration of the antibody which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography. In some embodiments, e.g., wherelocal administration (e.g., to the eye or a joint) is desired, cellculture or animal modeling can be used to determine a dose required toachieve a therapeutically effective concentration within the local site.

In some embodiments, the methods can be performed in conjunction withother therapies for cancer. For example, the composition can beadministered to a subject at the same time, prior to, or after,radiation, surgery, targeted or cytotoxic chemotherapy,chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, celltransplant therapy, precision medicine, genome editing therapy, or otherpharmacotherapy.

As described above, the compositions described herein (e.g., anti-CD137compositions) can be used to treat a variety of cancers such as but notlimited to: Kaposi's sarcoma, leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, myeloblasts promyelocyte myelomonocyticmonocytic erythroleukemia, chronic leukemia, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin'sdisease, non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors, sarcomas, andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma,osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular tumor, lung carcinoma, small celllung carcinoma, non-small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basalcell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brainand central nervous system (CNS) cancer, cervical cancer,choriocarcinoma, colorectal cancers, connective tissue cancer, cancer ofthe digestive system, endometrial cancer, esophageal cancer, eye cancer,head and neck cancer, gastric cancer, intraepithelial neoplasm, kidneycancer, larynx cancer, liver cancer, lung cancer (small cell, largecell), melanoma, neuroblastoma; oral cavity cancer (for example lip,tongue, mouth and pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of therespiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem.

In some embodiments, an anti-CD137 antibody or an antigen-bindingfragment thereof described herein can be administered to a subject as amonotherapy. Alternatively, as described above, the antibody or fragmentthereof can be administered to a subject as a combination therapy withanother treatment, e.g., another treatment for a cancer. For example,the combination therapy can include administering to the subject (e.g.,a human patient) one or more additional agents that provide atherapeutic benefit to a subject who has, or is at risk of developing,cancer. Chemotherapeutic agents suitable for co-administration withcompositions of the present invention include, for example: taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Further agents include, for example, antimetabolites(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine,thioTEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, cis-dichlordiamine platinum (II)(DDP), procarbazine, altretamine,cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, ortriplatin tetranitrate), anthracycline (e.g. daunorubicin (formerlydaunomycin) and doxorubicin), antibiotics (e.g. dactinomcin (formerlyactinomycin), bleomycin, mithramycin, and anthramycin (AMC)), andanti-mitotic agents (e.g. vincristine and vinblastine) and temozolomide.In some embodiments, an anti-CD137 antibody and the one or moreadditional active agents are administered at the same time. In otherembodiments, the anti-CD137 antibody is administered first in time andthe one or more additional active agents are administered second intime. In some embodiments, the one or more additional active agents areadministered first in time and the anti-CD137 antibody is administeredsecond in time.

An anti-CD137 antibody or an antigen-binding fragment thereof describedherein can replace or augment a previously or currently administeredtherapy. For example, upon treating with an anti-CD137 antibody orantigen-binding fragment thereof, administration of the one or moreadditional active agents can cease or diminish, e.g., be administered atlower levels or dosages. In some embodiments, administration of theprevious therapy can be maintained. In some embodiments, a previoustherapy will be maintained until the level of the anti-CD137 antibodyreaches a level sufficient to provide a therapeutic effect. The twotherapies can be administered in combination.

Monitoring a subject (e.g., a human patient) for an improvement in acancer, as defined herein, means evaluating the subject for a change ina disease parameter, e.g., a reduction in tumor growth. In someembodiments, the evaluation is performed at least one (1) hour, e.g., atleast 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration.The subject can be evaluated in one or more of the following periods:prior to beginning of treatment; during the treatment; or after one ormore elements of the treatment have been administered. Evaluation caninclude evaluating the need for further treatment, e.g., evaluatingwhether a dosage, frequency of administration, or duration of treatmentshould be altered. It can also include evaluating the need to add ordrop a selected therapeutic modality, e.g., adding or dropping any ofthe treatments for a cancer described herein.

In some embodiments, an anti-CD137 antibody or an antigen-bindingfragment thereof described herein is administered to modulate a T-cellresponse in a patient, for example, by increasing T-cell activationand/or proliferation. Crosslinking of CD137 strongly enhances T cellproliferation, IFNγ production and secretion, and the cytolytic activityof T cells. Accordingly, in some embodiments, an anti-CD137 agonistantibody, or an antigen-binding fragment thereof, of the presentdisclosure is administered to a patent in need thereof to induce orincrease T-cell activation, enhance T cell proliferation, induce theproduction and/or secretion of IFNγ, and/or induce a cytolytic T cellresponse.

In some embodiments, an anti-CD137 antibody or an antigen-bindingfragment thereof described herein is useful to modulate or shift theT-cell population in a patient from a T_(H)2/T_(reg) T cell populationto a T_(H)1/T_(H)17 T cell population to thereby improve or enhance ananti-tumor response in the patient. Studies have shown that while CD137is expressed in both T-cell subsets, Th1 and Th2 T cells, CD137 isexpressed at higher levels on CD8+ T cells than on CD4+ T cells.Accordingly, CD137 mainly co-stimulates CD8+ T cells. Accordingly, ananti-CD137 antibody, or an antigen-binding fragment thereof, asdescribed herein, is administered to a patient to enhance an anti-tumorresponse, for example, by modulating or shifting the T-cell responseand/or T cell population in the patient from a T_(H)2/T_(reg) T cellresponse and or T cell population to a T_(H)1/T_(H)17 T cell responseand/or T cell population in the patient.

In some cancers (e.g. melanoma and ovarian cancer), naturaltumor-infiltrating lymphocytes (TILs) can be enriched through optimizedcell culture methods and provide a source of tumor-reactive lymphocytesuseful for adoptive immunotherapy. Adoptive TIL therapy can result indurable tumor regression for some types of cancer, which warrants thedevelopment and optimization of TIL-based approaches for cancer.Currently, the identification and expansion of natural tumor-reactiveTILs remains challenging due to low level and/or rarity ofantigen-specific CD8+ T cells. CD137 expression by T cells is activationdependent, which provides an opportunity to capture CD137-expressingactivated T cells from circulation or from tumor samples. Accordingly,an anti-CD137 antibody, or an antigen-binding fragment thereof, asdescribed herein, can be employed for the selective enrichment ofactivated, antigen-specific T cells.

In some embodiments, the efficacy of the anti-CD137 antibodies describedherein is dependent on a competent immune system. Specifically, in someembodiments, depletion of CD4+ T cells, CD8+ T cells and/or NaturalKiller cells reduces the efficacy of the anti-CD137 antibodies. In someembodiments, depletion of CD4+ T cells, CD8+ T cells and/or NaturalKiller cells reduces the inhibition or reduction of tumor growth by theanti-CD137 antibodies described herein. In some embodiments, depletionof CD4+ T cells, CD8+ T cells and/or Natural Killer cells reduces theinhibition or reduction of tumor growth by the anti-CD137 antibodiesdescribed herein. In some embodiments, the efficacy of the anti-CD137antibodies described herein is dependent on an infiltration of immunecells into a tumor microenvironment. In some embodiments, theinfiltration of immune cells into a tumor microenvironment is coupledwith a lack of infiltration into the spleen and/or liver.

In some embodiments, the anti-CD137 antibodies described herein induce aprotective anti-tumor memory immune response. Memory T cells are asubset of antigen-specific T cells that persist long-term after havingencountered and responded to their cognate antigen. Such cells quicklyexpand to large numbers of effector cells upon re-exposure to theircognate antigen. Accordingly, in some embodiments the anti-CD137antibodies described herein stimulate the production of memory T cellsto a cancer antigen. In some embodiments, a subject that has received ananti-CD137 antibody described herein to treat or cure a cancer, developsmemory T cells specific to the cancer. In some embodiments, a subjectthat has received an anti-CD137 antibody described herein to treat orcure a cancer, develops an anti-tumor memory immune response uponre-exposure to the cancer. In some embodiments, the anti-tumor memoryimmune response comprises stimulating memory T cells to become effectorcells. In some embodiments, a subject that has received an anti-CD137antibody described herein to treat or cure a cancer, develops ananti-tumor memory immune response to a cancer antigen.

In some embodiments, the anti-CD137 antibodies described herein induceimmune re-programming with a tumor microenvironment. Specifically, insome embodiments, the anti-CD137 antibodies induce immune infiltration;reduce, inhibit or prevent Treg proliferation; reduce, inhibit orprevent tumor-associated macrophage proliferation; and protect orreverse T cell exhaustion.

In some embodiments, the anti-CD137 antibodies induce infiltration ofimmune cells into a tumor microenvironment relative. In someembodiments, the anti-CD137 antibodies increase immune cell infiltrationby at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 100%, atleast 105%, at least 110%, at least 115%, at least 120%, at least 125%,at least 130%, at least 135%, at least 140%, at least 145%, or at least150%. In some embodiments, immune cell infiltration is determined bymeasuring the level of CD45 expression on cells isolated from a tumormicroenvironment. Methods for measuring protein expression are known tothose of skill in the art and described herein.

In some embodiments, the anti-CD137 antibodies prevent or inhibit anincrease in Treg cells in a tumor microenvironment. In some embodiments,prevention or inhibition is relative to the amount of Treg cells in atumor microenvironment in the absence of an anti-CD137 antibody. In someembodiments, prevention or inhibition of an increase in Treg cells isrelative to a reference antibody. In some embodiments, Treg cells aredetected by expression of CD25 and FOX-3P on CD4+ T cells isolated froma tumor microenvironment. Methods for measuring protein expression areknown to those of skill in the art and described herein.

In some embodiments, the anti-CD137 antibodies prevent or inhibit anincrease in tumor-associated macrophages in a tumor microenvironment. Insome embodiments, prevention or inhibition is relative to the amount oftumor-associated macrophages in a tumor microenvironment in the absenceof an anti-CD137 antibody. In some embodiments, prevention or inhibitionof an increase in tumor-associated macrophages is relative to areference antibody. In some embodiments, tumor-associated macrophagesare detected by expression of CD1 lb and F4/80 on CD45+ immune cellsisolated from a tumor microenvironment. Methods for measuring proteinexpression are known to those of skill in the art and described herein.

In some embodiments, the anti-CD137 antibodies protect T cells from Tcell exhaustion in a tumor microenvironment. In some embodiments, theanti-CD137 antibodies reverse T cell exhaustion in a tumormicroenvironment. In some embodiments, T cell exhaustion in a tumormicroenvironment is reduced in the presence of an anti-CD137 antibodydescribed herein, relative to a tumor microenvironment in the absence ofthe anti-CD137 antibody. In some embodiments, T cell exhaustion isdetermined by analyzing CD8+ T cells or CD4+ T cells for expression ofco-inhibitory receptors (e.g., PD-1, TIGIT or LAG-3). In someembodiments, T cell exhaustion is detected by expression of PD-1 andTIGIT on CD4+ or CD8+ T cells isolated from a tumor microenvironment.

In some embodiments, an anti-CD137 antibody, or an antigen-bindingfragment thereof, described herein, can be employed in methods ofdetection and/or quantification of human CD137 in a biological sample.Accordingly, an anti-CD137 antibodies, or an antigen-binding fragmentthereof, as described herein, is used to diagnose, prognose, and/ordetermine progression of disease (e.g., cancer) in a patient.

OTHER EMBODIMENTS

E1. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain CDRs selectedfrom the group consisting of:

(a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 70, 79 and 90, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 71, 80 and 91, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 72, 81 and 92, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 73, 82 and 91, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 74, 83 and 93, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 75, 84 and 91, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 74, 85 and 94, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 76, 86 and 95, respectively;

(j) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 77, 87 and 93, respectively;

(k) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 88 and 90, respectively;

(l) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 57 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(m) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 58 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(n) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 59 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(o) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:49, 60 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(p) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 61 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(q) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 58 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(r) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 62 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(s) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:52, 63 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(t) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 64 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(u) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:50, 65 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(v) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 108 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively;

(w) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:107, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively; and

(x) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 109, 110 and 92, respectively.

E2. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain variableregions, wherein the heavy chain variable region comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 101 and 103; and wherein the light chainvariable region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and105.E3. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain CDRs, whereinheavy chain CDR3 comprises the amino acid sequence set forth in SEQ IDNO: 68.E4. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain variable regionscomprising amino acid sequences selected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively;

(b) SEQ ID NO: 4 and 28, respectively;

(c) SEQ ID NO: 4 and 30, respectively;

(d) SEQ ID NO: 4 and 32, respectively;

(e) SEQ ID NO: 4 and 34, respectively;

(f) SEQ ID NO: 4 and 36, respectively;

(g) SEQ ID NO: 4 and 38, respectively;

(h) SEQ ID NO: 4 and 40, respectively;

(i) SEQ ID NO: 4 and 42, respectively;

(j) SEQ ID NO: 4 and 44, respectively;

(k) SEQ ID NO: 4 and 46, respectively;

(l) SEQ ID NO: 8 and 6, respectively;

(m) SEQ ID NO: 10 and 6, respectively;

(n) SEQ ID NO: 12 and 6, respectively;

(o) SEQ ID NO: 14 and 6, respectively;

(p) SEQ ID NO: 16 and 6, respectively;

(q) SEQ ID NO: 18 and 6, respectively;

(r) SEQ ID NO: 20 and 6, respectively;

(s) SEQ ID NO: 22 and 6, respectively;

(t) SEQ ID NO: 24 and 6, respectively;

(u) SEQ ID NO: 26 and 6, respectively;

(v) SEQ ID NO: 101 and 6, respectively;

(w) SEQ ID NO: 103 and 6, respectively; and

(x) SEQ ID NO: 4 and 105, respectively.

E5. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain variableregions, wherein the heavy chain variable region comprises an amino acidsequence which is at least 90% identical to the amino acid sequenceselected from the group consisting of SEQ ID NOs: 4, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 101 and 103; and wherein the light chain variableregion comprises an amino acid sequence which is at least 90% identicalto the amino acid sequence selected from the group consisting of SEQ IDNOs: 6, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 105.E6. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain variable regionscomprising amino acid sequences at least 90% identical to the amino acidsequences selected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively;

(b) SEQ ID NO: 4 and 28, respectively;

(c) SEQ ID NO: 4 and 30, respectively;

(d) SEQ ID NO: 4 and 32, respectively;

(e) SEQ ID NO: 4 and 34, respectively;

(f) SEQ ID NO: 4 and 36, respectively;

(g) SEQ ID NO: 4 and 38, respectively;

(h) SEQ ID NO: 4 and 40, respectively;

(i) SEQ ID NO: 4 and 42, respectively;

(j) SEQ ID NO: 4 and 44, respectively;

(k) SEQ ID NO: 4 and 46, respectively;

(l) SEQ ID NO: 8 and 6, respectively;

(m) SEQ ID NO: 10 and 6, respectively;

(n) SEQ ID NO: 12 and 6, respectively;

(o) SEQ ID NO: 14 and 6, respectively;

(p) SEQ ID NO: 16 and 6, respectively;

(q) SEQ ID NO: 18 and 6, respectively;

(r) SEQ ID NO: 20 and 6, respectively;

(s) SEQ ID NO: 22 and 6, respectively;

(t) SEQ ID NO: 24 and 6, respectively;

(u) SEQ ID NO: 26 and 6, respectively;

(v) SEQ ID NO: 101 and 6, respectively;

(w) SEQ ID NO: 103 and 6, respectively; and

(x) SEQ ID NO: 4 and 105, respectively.

E7. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain CDRs, whereinheavy chain CDR3 comprises the amino acid sequence set forth in SEQ IDNO: 68.E8. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain CDRs, whereinheavy chain CDR3 comprises the amino acid sequence DXXXXLXXXXYXYYX (SEQID NO: 126), wherein X is any amino acid.E9. An isolated monoclonal antibody that specifically binds human CD137,or antigen binding portion thereof, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain CDRs, whereinheavy chain CDR3 comprises the amino acid sequence DXPFXLDXXYYYYYX (SEQID NO: 127), wherein X is any amino acid.E10. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein the antibody orantigen binding portion thereof comprises heavy and light chain CDRs,wherein heavy chain CDR3 comprises the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid except foralanine.E11. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein the antibody orantigen binding portion thereof comprises heavy and light chain CDRs,wherein heavy chain CDR3 comprises the amino acid sequenceDXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X is any amino acid except foralanine.E12. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein the antibody orantigen binding portion thereof comprises heavy and light chain CDRs,wherein heavy chain CDR3 comprises the amino acid sequenceDXXXXLXXXXYXYYX (SEQ ID NO: 126), wherein X is any amino acid, andwherein mutation of residues D95, L100, Y100E, Y100G, Y100H, orcombinations thereof, results in loss of binding to human CD137.E13. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein the antibody orantigen binding portion thereof comprises heavy and light chain CDRs,wherein heavy chain CDR3 comprises the amino acid sequenceDXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X is any amino acid, andwherein mutation of residues P97, F98, D100A, Y100D, Y100F, orcombinations thereof to alanine results in reduction of binding to humanCD137.E14. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein the antibody orantigen binding portion thereof comprises heavy and light chain CDRs,wherein heavy chain CDR3 comprises the amino acid sequenceDXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X is any amino acid, andwherein mutation of residues P97, F98, D100A, Y100D, Y100F, orcombinations thereof to any residue except alanine, results in anincrease in binding to human CD137.E15. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein the antibody orantigen binding portion thereof comprises heavy and light chain CDRs,wherein heavy chain CDR3 comprises the amino acid sequenceDX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQ ID NO: 128), wherein X₁ is any aminoacid, wherein X₂ is a non-polar amino acid, wherein X₃ is a non-polaramino acid, wherein X₄ is any amino acid, wherein X₅ is a polar aminoacid, wherein X₆ is any amino acid, wherein X₇ is any amino acid,wherein X₈ is a polar amino acid, wherein X₉ is a polar amino acid, andwherein X₁₀ is any amino acid.E16. The isolated monoclonal antibody of embodiment 15, wherein X₂ isproline, wherein X₃ is phenylalanine or tryptophan, wherein X₅ isaspartic acid or glutamic acid, wherein X₈ is tyrosine, and wherein X₉is tyrosine.E17. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 8-16, wherein the antibody or antigen bindingportion thereof cross competes with mAb1.E18. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 8-16, wherein the antibody or antigen bindingportion thereof cross competes with mAb1, mAb8 or mAb10.E19. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 8-18, wherein the antibody or antigen bindingportion thereof comprises at least the functional properties of mAb1.E20. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 8-18, wherein the antibody or antigen bindingportion thereof comprises at least the functional properties of mAb1,mAb8 or mAb10.E21. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 8-20, wherein the antibody or antigen bindingportion thereof has a K_(D) value at least equivalent to mAb1.E22. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 8-20, wherein the antibody or antigen bindingportion thereof has a K_(D) value at least equivalent to mAb1, mAb8 ormAb10.E23. An isolated monoclonal antibody, or antigen binding portionthereof, that specifically binds to human CD137, wherein, when bound tohuman CD137, the isolated monoclonal antibody, or antigen bindingportion thereof, binds to at least one of the amino acid residues boundby mAb1, or an antigen binding fragment of mAb1.E24. An isolated monoclonal antibody, or antigen binding portionthereof, that specifically binds to human CD137, wherein, when bound tohuman CD137, the isolated monoclonal antibody, or antigen bindingportion thereof: (i) binds to at least one of the amino acid residuesbound by mAb1, or an antigen binding fragment of mAb1, and (ii) agonizeshuman CD137.E25. The isolated monoclonal antibody, or antigen binding portionthereof, of any one of embodiments 23-24, wherein the amino acidresidues comprising the epitope bound by the antibody are located within4 angstroms of the amino acid residues comprising the paratope of themAb1 antibody.E26. The isolated monoclonal antibody, or antigen binding portionthereof, of any one of the embodiments 23-25, wherein a mutation of theepitope bound by the antibody inhibits, reduces, or blocks binding toboth the antibody and to antibody mAb1.E27. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein the antibody orantigen binding portion binds human CD137 with an affinity (K_(D)) ofabout 40-100 nM.E28. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 40-100 nM; and

(ii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DXXXXLXXXXYXYYX (SEQ ID NO:126), wherein X is any amino acid.

E29. An isolated monoclonal antibody that specifically binds humanCD137, or antigen binding portion thereof, wherein

(i) the antibody or antigen binding portion binds human CD137 with anaffinity (K_(D)) of about 40-100 nM; and

(ii) the antibody or antigen binding portion comprises a heavy chainCDR3 comprising the amino acid sequence DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ (SEQID NO: 128), wherein X₁ is any amino acid, wherein X₂ is a non-polaramino acid, wherein X₃ is a non-polar amino acid, wherein X₄ is anyamino acid, wherein X₅ is a polar amino acid, wherein X₆ is any aminoacid, wherein X₇ is any amino acid, wherein X₈ is a polar amino acid,wherein X₉ is a polar amino acid, and wherein X₁₀ is any amino acid.

E30. The isolated monoclonal antibody or antigen binding portion thereofof embodiment 27, wherein the antibody or antigen binding portionthereof comprises a heavy chain CDR3 comprising the amino acid sequenceDXPFXLDXXYYYYYX (SEQ ID NO: 127), wherein X is any amino acid.E31. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 28-30, wherein mutation of residues D95, L100,Y100E, Y100G, Y100H, or combinations thereof, of the heavy chain CDR3,results in loss of binding to human CD137.E32. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 28-31, wherein mutation of residues P97, F98,D100A, Y100D, Y100F, or combinations thereof to alanine results inreduction of binding to human CD137.E33. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 28-31, wherein mutation of residues P97, F98,D100A, Y100D, Y100F, or combinations thereof to any residue exceptalanine, results in an increase in binding to human CD137.E34. The isolated monoclonal antibody or antigen binding portionthereof, of any one of embodiments 28 and 29-33, wherein X is any aminoacid except for alanine.E35. The isolated monoclonal antibody or antigen binding portionthereof, of any one of embodiments 29, and 31-33, wherein X₂ is proline,wherein X₃ is phenylalanine or tryptophan, wherein X₅ is aspartic acidor glutamic acid wherein X₈ is tyrosine, and wherein X₉ is tyrosineE36. The isolated monoclonal antibody or antigen binding portion thereofof any one of the preceding embodiments, wherein the antibody or antigenbinding portion binds human CD137 with an affinity (K_(D)) of about45-95 nM, 50-90 nM, 55-85 nM, 60-80 nM, 65-75 nM, 55-75 nM, 40-70 nM,50-80 nM, or 60-90 nM.E37. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 27-36, wherein the antibody or antigen bindingportion thereof comprises heavy and light chain CDRs, wherein heavychain CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68.E38. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 27-37, wherein the antibody or antigen bindingportion thereof comprises heavy and light chain CDRs selected from thegroup consisting of:

(a) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:48, 56 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively; and

(b) heavy chain CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NOs:51, 108 and 68, respectively, and light chain CDR1, CDR2 and CDR3sequences set forth in SEQ ID NOs: 69, 78 and 89, respectively.

E39. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 27-37, wherein the antibody or antigen bindingportion thereof comprises heavy and light chain variable regions,wherein the heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 4 and 101; and whereinthe light chain variable region comprises an amino acid sequence of SEQID NO: 6.E40. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 27-37, wherein the antibody or antigen bindingportion thereof comprises heavy and light chain variable regionscomprising amino acid sequences selected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively; and

(b) SEQ ID NO: 101 and 6, respectively.

E41. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 27-37, wherein the antibody or antigen bindingportion thereof comprises heavy and light chain variable regions,wherein the heavy chain variable region comprises an amino acid sequencewhich is at least 90% identical to the amino acid sequence selected fromthe group consisting of SEQ ID NOs: 4 and 101; and wherein the lightchain variable region comprises an amino acid sequence which is at least90% identical to the amino acid sequence of SEQ ID NO: 6.E42. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 27-37, wherein the antibody or antigen bindingportion thereof comprises heavy and light chain variable regionscomprising amino acid sequences at least 90% identical to the amino acidsequences selected from the group consisting of:

(a) SEQ ID NO: 4 and 6, respectively; and

(b) SEQ ID NO: 101 and 6, respectively.

E43. The isolated monoclonal antibody, or antigen binding portionthereof of any one of the preceding embodiments, wherein the antibody orantigen binding portion thereof specifically binds to and agonizes humanCD137.

E44. The isolated monoclonal antibody or antigen binding portion thereofof any one of the preceding embodiments, wherein the antibody or antigenbinding portion thereof exhibits at least one or more of the followingproperties:

(a) induces or enhances dimerization of CD137 trimers;

(b) induces or enhances multimerization of CD137 trimers;

(c) induces or enhances human CD137-mediated T cell activation;

(d) induces or enhances a human CD137-mediated cytotoxic T cellresponse;

(e) induces or enhances human CD137-mediated T cell proliferation;

(f) induces or enhances human CD137-mediated cytokine production;

(g) does not significantly induce or enhance intrahepatic and/orintrasplenic T cell activation and/or T cell proliferation;

(h) binds to human CD137 with an equilibrium dissociation constant K_(D)of 1×10⁻⁶ or less; or

(i) any combination of properties (a)-(h).

E45. The isolated monoclonal antibody or antigen binding portion thereofof embodiment 44, wherein the antibody or antigen binding portionthereof induces or enhances human CD137-mediated T cell activation inthe tumor microenvironment, but does not significantly induce or enhancehuman CD137-mediated T cell activation in the spleen and/or liver.E46. The isolated monoclonal antibody or antigen binding portion thereofof embodiment 44, wherein the antibody or antigen binding portionthereof induces or enhances human CD137-mediated cytotoxic T cellresponse in the tumor microenvironment, but does not significantlyinduce or enhance human CD137-mediated cytotoxic T cell response in thespleen and/or liver.E47. The isolated monoclonal antibody or antigen binding portion thereofof embodiment 44, wherein the antibody or antigen binding portionthereof induces human CD137-mediated T cell proliferation in the tumormicroenvironment, but does not significantly induce human CD137-mediatedT cell proliferation in the spleen and/or liver.E48. The isolated monoclonal antibody or antigen binding portion thereofof embodiment 44, wherein the antibody or antigen binding fragmentthereof induces or enhances human CD137-mediated cytokine production inthe tumor microenvironment, but does not significantly induce or enhancehuman CD137-mediated cytokine production in the spleen and/or liver.E49. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 44-48, wherein the properties of the antibodyor antigen binding portion thereof are not Fc receptor bindingdependent.E50. The isolated monoclonal antibody or antigen binding portion thereofof any one of embodiments 44-49, wherein the properties of the antibodyor antigen binding portion thereof are enhanced by Fc receptor binding.E51. The isolated monoclonal antibody, or antigen binding portionthereof, of any one of the preceding embodiments, wherein the antibodyor antigen binding portion thereof cross-reacts with cynomolgus CD137and/or mouse CD137.E52. An agonistic isolated monoclonal antibody that binds to human CD137and exhibits at least one of the following properties:

(a) induces or enhances dimerization of human CD137 trimers;

(b) induces or enhances multimerization of human CD137 trimers;

(c) induces or enhances human CD137-mediated T cell activation in thetumor microenvironment, but does not significantly induce or enhancehuman CD137-mediated T cell activation in the spleen and/or liver;

(d) induces or enhances a human CD137-mediated cytotoxic T cell responsein the tumor microenvironment, but does not significantly induce orenhance human CD137-mediated cytotoxic T cell response in the spleenand/or liver;

(e) induces or enhances human CD137-mediated cytokine production in thetumor microenvironment, but does not significantly induce or enhancehuman CD137-mediated cytokine production in the spleen and/or liver;

(f) induces or enhances human CD137-mediated T cell proliferation in thetumor microenvironment, but does not significantly induce or enhancehuman CD137-mediated T cell proliferation in the spleen and/or liver;

(g) binds to human CD137 with an equilibrium dissociation constant K_(D)of 1×10⁻⁶ or less; or

(h) any combination of properties (a)-(g).

E53. The isolated monoclonal antibody, or antigen binding portionthereof, according to any one of the preceding embodiments, wherein theantibody is selected from the group consisting of an IgG1, an IgG2, andIgG3, an IgG4, and IgM, and IgA1, and IgA2, and IgD, and an IgEantibody.E54. The isolated monoclonal antibody, or antigen binding portionthereof, of embodiment 53, wherein the antibody is an IgG1 antibody orIgG4 antibody.E55. A pharmaceutical composition comprising an isolated monoclonalantibody or antigen binding portion thereof, of any one of the precedingembodiments, and a pharmaceutically acceptable carrier.E56. A nucleic acid comprising a nucleotide sequence encoding the lightchain, heavy chain, or both light and heavy chains of the isolatedmonoclonal antibody, or antigen binding portion thereof, of any one ofembodiments 1-54.E57. An expression vector comprising the nucleic acid of embodiment 56.E58. A cell transformed with an expression vector of embodiment 57.E59. A method for producing a monoclonal antibody that specificallybinds human CD137, or an antigen binding portion thereof, the methodcomprising maintaining a cell according to embodiment 58 underconditions permitting expression of the monoclonal antibody or antigenbinding portion thereof.E60. The method of embodiment 59, further comprising obtaining themonoclonal antibody or antigen binding portion thereof.E61. A method for inducing or enhancing dimerization of human CD137trimers in a subject, comprising administering to a subject in needthereof, an effective amount of an isolated monoclonal antibody, orantigen binding portion thereof, of any one of embodiments 1-54, or thepharmaceutical composition of embodiment 55.E62. A method for inducing or enhancing multimerization of human CD137trimers in a subject, comprising administering to a subject in needthereof, an effective amount of an isolated monoclonal antibody, orantigen binding portion thereof, of any one of embodiments 1-54, or thepharmaceutical composition of embodiment 55.E63. A method for inducing or enhancing T cell activation mediated byhuman CD137 in a subject, comprising administering to a subject in needthereof, an effective amount of an isolated monoclonal antibody, orantigen binding portion thereof, of any one of embodiments 1-54, or thepharmaceutical composition of embodiment 55.E64. The method of embodiment 63, wherein the T cell activation occursin a tumor microenvironment.E65. The method of embodiment 63, wherein the T cell activation does notsignificantly occur in the spleen and/or liver of the subject.E66. A method for inducing or enhancing a cytotoxic T cell responsemediated by human CD137 in a subject, comprising administering to asubject in need thereof, an effective amount of an isolated monoclonalantibody, or antigen binding portion thereof, of any one of embodiments1-54, or the pharmaceutical composition of embodiment 55.E67. The method of embodiment 66, wherein the cytotoxic T cell responseoccurs in a tumor microenvironment.E68. The method of embodiment 66, wherein the cytotoxic T cell responsedoes not significantly occur in the spleen and/or liver of the subject.E69. A method for inducing or enhancing cytokine production mediated byhuman CD137 in a subject, comprising administering to a subject in needthereof, an effective amount of an isolated monoclonal antibody, orantigen binding portion thereof, of any one of embodiments 1-54, or thepharmaceutical composition of embodiment 55.E70. The method of embodiment 69, wherein the cytokine produced is IL-2,TNFα, IL-13, IFNγ, or combinations thereof.E71. The method of embodiment 69 or embodiment 70, wherein the cytokineproduction occurs in a tumor microenvironment.E72. The method of embodiment 69 or embodiment 70, wherein the cytokineproduction does not significantly occur in the spleen and/or liver ofthe subject.E73. A method for inducing or enhancing T cell proliferation mediated byhuman CD137 in a subject, comprising administering to a subject in needthereof, an effective amount of an isolated monoclonal antibody, orantigen binding portion thereof, of any one of embodiments 1-54, or thepharmaceutical composition of embodiment 55.E74. The method of embodiment 73, wherein the T cell proliferationoccurs in a tumor microenvironment.E75. The method of embodiment 73, wherein the T cell proliferation doesnot significantly occur in the spleen and/or liver of the subject.E76. A method for reducing or inhibiting tumor growth, comprisingadministering to a subject in need thereof, an effective amount of anisolated monoclonal antibody, or antigen binding portion thereof, of anyone of embodiments 1-54, or the pharmaceutical composition of embodiment55.E77. A method for treating a disorder mediated by human CD137 in asubject, comprising administering to a subject in need thereof, aneffective amount of an isolated monoclonal antibody, or antigen bindingportion thereof, of any one of embodiments 1-54, or the pharmaceuticalcomposition of embodiment 55.E78. A method for treating cancer in a subject, comprising administeringto a subject in need thereof, an effective amount of an isolatedmonoclonal antibody, or antigen binding portion thereof, of any one ofembodiments 1-54, or the pharmaceutical composition of embodiment 55.E79. The method of embodiment 78, wherein the cancer is selected fromthe group consisting of melanoma, glioma, renal, and head and neckcancer.E80. The method of any one of embodiments 76-79, wherein the antibody orantigen binding portion thereof binds Fc gamma receptor.E81. The method of any one of embodiments 76-80, wherein depletion ofCD4+ T cells, CD8+ T cells, Natural Killer cells, or combinationsthereof, reduces the efficacy of the antibody or antigen binding portionthereof.E82. A method for detecting the presence or absence of human CD137 in abiological sample, comprising:

(i) contacting a biological sample with the antibody of any one ofembodiments 1-54, wherein the antibody is labeled with a detectablesubstance; and

(ii) detecting the antibody bound to human CD137 to thereby detect thepresence or absence of human CD137 in the biological sample.

EXAMPLES

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

Example 1: Synthetic Human Monoclonal Antibodies Produced in YeastExhibit Binding to Recombinant Human CD137

Purified CD137 protein antigen was biotinylated using the EZ-LinkSulfo-NHS-Biotinylation Kit (Thermo Scientific). CD137 antigens wereconcentrated to ˜1 mg/mL and buffer exchanged into PBS before additionof 1:7.5 molar ratio biotinylation reagent (EZ-LinkSulfo-NHS-Biotinylation Kit, Thermo Scientific, Cat #21425.). Themixture was held at 4° C. overnight prior to another buffer exchange toremove free biotin in the solution. Biotinylation was confirmed throughStreptavidin sensor binding of the labeled proteins on a ForteBio.Successful biotinylation of the CD137 protein antigen was confirmed viadetectable binding to a streptavidin-linked biosensor installed onForteBio Octet™ Red384 Interferometer (Pall ForteBio, Menlo Park,Calif.) according to the manufacturer's guidelines (data not shown).

Eight naïve human synthetic yeast-based antibody libraries each of ˜10⁹diversity were designed, generated, and propagated as describedpreviously (see, e.g., WO2009036379; WO2010105256, WO2012009568; Xu etal., Protein Eng Des Sel. 2013 October; 26(10):663-70). Eight parallelselections were performed, using the eight naïve libraries againstbiotinylated human CD137-Fc fusion.

For the first two rounds of selection, a magnetic bead sorting techniqueutilizing the Miltenyi MACS system was performed, essentially asdescribed (Siegel et al., J Immunol Methods. 2004 March;286(1-2):141-53). Briefly, yeast cells (˜10¹⁰ cells/library) wereincubated with 10 mL of 10 nM biotinylated human CD137-Fc fusion antigenfor 15 minutes at room temperature in FACS wash buffer PBS with 0.1%BSA. After washing once with 50 mL ice-cold wash buffer, the cell pelletwas resuspended in 40 mL wash buffer, and 500 μl Streptavidin MicroBeads(Miltenyi Biotec, Bergisch Gladbach, Germany. Cat #130-048-101) wereadded to the yeast and incubated for 15 minutes at 4° C. Next, the yeastwere pelleted, resuspended in 5 mL wash buffer, and loaded onto a MACSLS column (Miltenyi Biotec, Bergisch Gladbach, Germany.Cat.#130-042-401). After the 5 mL was loaded, the column was washedthree times with 3 mL FACS wash buffer. The column was then removed fromthe magnetic field, and the yeast were eluted with 5 mL of growth mediaand then grown overnight.

Subsequent to the two rounds of MACS, three rounds of sorting wereperformed using flow cytometry (FACS), which are described in thefollowing three paragraphs.

Selection Strategy Employing 8 Parallel Selections with Fc Antigen

The eight libraries from the MACS selections were taken through threerounds of FACS selections. Approximately 1×10⁸ yeast per library werepelleted, washed three times with wash buffer, and incubated with 10 nMof biotinylated human CD137-Fc fusion and 10 nM of biotinylated murineCD137-Fc fusion antigen separately for 10 minutes at room temperature.Yeast were then washed twice and stained with goat anti-human F(ab′) 2kappa-FITC diluted 1:100 (Southern Biotech, Birmingham, Ala.,Cat#2062-02) and either streptavidin-Alexa Fluor 633 (Life Technologies,Grand Island, N.Y., Cat # S21375) diluted 1:500, orExtravidin-phycoerthyrin (Sigma-Aldrich, St Louis, Cat # E4011) diluted1:50, secondary reagents for 15 minutes at 4° C. After washing twicewith ice-cold wash buffer, the cell pellets were resuspended in 0.4 mLwash buffer and transferred to strainer-capped sort tubes. Sorting wasperformed using a FACS ARIA sorter (BD Biosciences) and sort gates weredetermined to select only CD137 binding. The murine- and human-selectedpopulations from the first round of FACS were brought forward into thenext round.

The second and third round of FACS for the above selected populationsinvolved positive sorts for binders to human and/or murine CD137reagents; or negative sorts to decrease polyspecific reagent binders (Xuet al., PEDS. 2013 October; 26(10):663-70). Depending on the amount ofpolyspecific binding or target binding of a specific selection output, apositive sort followed a negative sort or vice versa, to enrich for afull binding population with limited amount of polyspecific binding.Competition selections were also performed with control mAbs from theliterature. For competition selections, mAb4 (urelumab; Bristol-MyersSquibb; CAS Number: 934823-49-1) and mAb5 (utomilumab; Pfizer; CASNumber: 1417318-27-4) were pre-complexed to biotinylated human CD137-Fcfusion. Antibodies that bind and do not bind in the presence of thecontrol mAbs were selected for on FACS. The outputs of these rounds wereplated and isolates were picked for sequencing and characterization.

Affinity Maturation of Clones Identified in Naïve Selections

Heavy chains from the first FACS sorting round against biotinylatedhuman CD137 Fc fusion outputs were used to prepare light chaindiversification libraries used for four additional selection rounds. Thefirst of these selection rounds utilized Miltenyi MACs beads conjugatedwith 10 nM biotinylated human CD137-Fc fusion as antigen.

Subsequent to the MACs bead selections, three rounds of FACS sortingwere performed. The first of these rounds used biotinylated humanCD137-Fc fusion at 10 nM. The second FACS round for the above involvedpositive sorts for binders to mouse CD137 reagents, competition sortswith previously mentioned control mAbs or negative sorts to decreasepolyspecific reagent binders as described above. The third and finalround of FACS selection was done using either biotinylated murine CD137Fc fusion at 10 nM or biotinylated human monomeric CD137 at 50 nM.Individual colonies from each FACS selection round described above werepicked for sequencing characterization.

IgG and Fab Production and Purification

Yeast clones were grown to saturation and then induced for 48 hours at30° C. with shaking. After induction, yeast cells were pelleted and thesupernatants were harvested for purification. IgGs were purified using aProtein A column and eluted with acetic acid, pH 2.0. Fab fragments weregenerated by papain digestion and purified over CaptureSelect IgG-CH1affinity matrix (LifeTechnologies, Cat #1943200250).

Example 2: Epitope Binning and Determination of Human Anti-CD137Antibody Affinity to Recombinant CD137

Epitope binning of the antibodies isolated in Example 1 was performed ona Forte Bio Octet Red384 system (Pall Forte Bio Corporation, Menlo Park,Calif.) using a standard sandwich format binning assay. CD137 controlantibody IgGs were loaded onto AHQ sensors and unoccupied Fc-bindingsites on the sensor were blocked with a non-relevant human IgG1antibody. The sensors were then exposed to 100 nM target antigenfollowed by exposure to the isolated antibodies identified as describedin Example 1. Data were processed using ForteBio's Data AnalysisSoftware 7.0. Additional binding by the second antibody after antigenassociation indicates an unoccupied epitope (non-competitor), while nobinding indicates epitope blocking (competitor) (data not shown).

Affinity of the CD137 antibodies was determined by measuring theirkinetic constants (k_(a), k_(d), K_(D)) on ForteBio Octet. ForteBioaffinity measurements were performed generally as previously described(Estep et al., MAbs. 2013 5(2):270-8). Briefly, ForteBio affinitymeasurements were performed by loading antibodies (IgGs) on-line ontoAHQ sensors. Sensors were equilibrated off-line in assay buffer for 30minutes and then monitored on-line for 60 seconds for baselineestablishment. For avid binding measurement, sensors with loaded IgGswere exposed to 100 nM antigen (human, cyno, or murine CD137) for 3minutes, afterwards they were transferred to assay buffer for 3 minutesfor off-rate measurement. Monovalent binding measurements were obtainedby loading human CD137-Fc fusion on AHQ sensors followed by exposure to200 nM antibody Fab in solution. Kinetics data were fit using a 1:1binding model in the data analysis software provided by ForteBio (datanot shown).

Determination of whether antibodies were ligand blocking was alsoassessed. Specifically, ligand blocking experiments were performed bothon Octet HTX (ForteBio) and on label-free MX96 SPRi (Caterra). mAb1 wascaptured on Octet sensor or MX96 chip sensor. CD137 and CD137L weresequentially applied to the sensors pre-loaded with mAb1. An increase inresponse upon exposure to CD137L indicated non-competition between mAb1and CD137L for binding to CD137. On the other hand, a lack of change inthe signal indicated competition, which was the case for controlantibody mAb5. mAb1 did not inhibit binding of CD137L to CD137 (data notshow), and therefore was considered a non-ligand blocking antibody.

Example 3: Distribution of Binding Affinities of Affinity-MaturedAnti-CD137 Antibodies

Affinity matured anti-CD137 antibodies were generated using 2 mutantlibraries. The first library contained mutations in the heavy chain andthe second library contained mutations in the light chain, wherein donordiversity in light chain CDR1, CDR2 and CDR3 was created. The mutantlibraries went through 3 rounds of phage panning aimed at increasingaffinity and maintaining cross-reactivity with mouse CD137. In eachround, an off-rate competition step was employed after initial bindingto biotinylated antigens (i.e., 1 hour incubation with excess unlabeledantigen or parental IgG at 37° C.).

The resulting anti-CD137 antibodies from different selection rounds wereplotted on k_(d)/k_(a) double log plots. Apparent association anddissociation kinetic rate constants (k_(a) and k_(d) values) weredetermined on an SPRi reader (MX96, Carterra) in a running buffer ofPBS-T 0.01%. Anti-human CD137 antibodies were covalently printed on aCarboxymethyldextran hydrogel 50 L chip (Xantec bioanalytics) on a CFM(Carterra). Freshly mixed activating reagents (150 ml 0.4 M EDC and 150ml 0.1 M sulfo-NHS in H2O) were used to activate the surface of the SPRsubstrate for 7 minutes. Antibodies at 10 mg/ml in acetic acid buffer pH4.5 were used for printing for 15 minutes. The printed chip was thenquenched on SPRi reader (MX96, Carterra) with 1 M ethanolamine for 15minutes. For kinetics analysis, purified recombinant his tagged humanCD137 (0, 2.05, 5.12, 12.8, 32, 80, 200, 500 nM) was injectedsequentially. For each concentration, there was 5 minutes of associationfollowed by 10 minutes of dissociation. Data were processed and analyzedin SPR Inspection Tool and Scrubber softwares. The kinetic data werereferenced with the interstitial reference spots and double-referencedto a buffer cycle, and then fit globally to a 1:1 binding model todetermine their apparent association and dissociation kinetic rateconstants (k_(a) and k_(d) values). The ratio k_(d)/k_(a) was used toderive the K_(D) value of each antigen/mAb interaction, i.e.K_(D)=k_(d)/k_(a).

Antibodies with K_(D) (k_(d)/k_(a)) between 10-20 nM are shown asupright triangles, while the ones with K_(D) lower than 10 nM are shownas upside down triangles (FIG. 1). Affinity maturation of only the heavychains (top panels) or only the light chains (bottom panels) bothresulted in the isolation of anti-CD137 antibodies with higher bindingaffinities than the parental antibody (mAb1) (FIG. 1). The heavy chainand light chain variable regions of mAb1 are set forth in SEQ ID NOs: 4and 6, respectively.

Example 4: Identification of Critical Binding Residues Comprising HeavyChain CDR3 (CDRH3) of Anti-CD137 Antibodies

To determine which amino acid residues within CDRH3 are critical for thebinding of mAb1 to mouse and human CD137 polypeptides, alanine scanningwas performed. A set of polynucleotides encoding derivatives of the mAb1open reading frame was generated, wherein each derivative contained asingle alanine residue substitution at a wild-type amino acid residueposition comprising CDRH3. Positions D95 through M100I of SEQ ID NO: 4were each mutated to alanine by replacing the wild-type codon with thealanine codon GCC. The amino acid sequences of each CDRH3 of each mAb1alanine-substituted derivative are set forth in SEQ ID NOs: 111-125. Thepolynucleotides encoding each of the 15 mAb1 alanine-substitutedderivatives were individually cloned into an expression vector(aglyco-IgG1, DID-2600) via Gibson Assembly. Each mAb1alanine-substituted derivative was expressed and purified using standardtechniques known in the art. Binding affinities of each mAb1alanine-substituted derivative for human and mouse CD137 were determinedvia Wasatch SPR kinetics measurements for human CD137 (huCD137) orequilibrium cell-binding assays for mouse CD137 (mCD137).

Table 1 provides the calculated dissociation constants (K_(D)) for eachmutant. When “Weak” is noted in the table there was measurable bindingabove background but not enough confidence in the curve fitting toassign an accurate K_(D) value. In Table 1, “NB” signifies that nobinding was observed during the determination of binding affinities andindicates which alanine substitutions in CDRH3 resulted in an antibodythat did not bind to CD137.

TABLE 1 Binding affinity (K_(D)) of alanine scanning clones for humanand mouse CD137 Substitution huCD137 mCD137 D95A NB NB S96A 1.8 nM 40 nMP97A Weak Weak F98A Weak Weak L99A 2.7 nM 33 nM L100A NB NB D(100A)AWeak Weak D(100B)A 1.3 nM 54 nM Y(100C)A   1 nM 25 nM Y(100D)A Weak 170nM  Y(100E)A NB NB Y(100F)A Weak Weak Y(100G)A NB NB Y(100H)A NB NBM(100I)A 1.8 nM 21 nM WT K_(D)   1 nM 11 nM

The retention, weakening, or loss of binding affinity resulting frommutations to alanine informed the determination of which residues wererequired for CD137 binding and which residues tolerated mutations. FIG.2 summarizes the binding data for alanine scanning of CDRH3 withwild-type amino acid identity indicated at each position. CDRH3positions are color-coded based on the effects of mutating the positionto alanine, as shown. This analysis resulted in the following consensussequence: DXPFXLDXXYYYYYX (SEQ ID NO: 127). When bolded residues in theconsensus sequence were mutated to alanine there was a complete loss ofbinding and these residues were therefore necessary for mAb1 binding toCD137. When italicized residues in the consensus sequence were mutatedto alanine the antibody was still able to bind CD137 but with a weakeraffinity indicating these residues played a partial role in binding butwere not absolutely required. When residue positions denoted with an Xin the consensus sequence were mutated to alanine there was little to nochange in binding affinity. Thus, these residues tolerated mutations andwere not critical to the binding interaction.

Example 5: Epitope Mapping by Scanning Saturation Mutagenesis andHomolog Comparison

Functional mapping of the CD137 epitope by scanning saturationmutagenesis library and homology comparison were performed to identifyresidues important for antibody binding to CD137. Combinatoriallibraries of CD137 mutants with single point mutations at all residuepositions to every possible amino acid substitution except cysteine weregenerated and tested for their ability to bind to mAb1, mAb4, and mAb5.A library consisting of genes encoding each point mutant of CD137 weresynthesized from a commercial supplier and cloned into a mammaliandisplay expression vector. Mammalian display was used to present alibrary of variant human CD137 extracellular domains, with each varianthaving at least one point mutation relative to wild type human CD137.

The library of cells displaying CD137 variants was stained withnon-overlapping antibodies (i) mAb4 and mAb1 or (ii) mAb4 and mAb5.Populations of cells with reduced binding to one antibody but not theother were enriched by FACS. Each population was sequenced by Illuminasequencing to identify mutations in positions that specificallydisrupted binding to each antibody but did not affect correct folding ofCD137 or binding to the non-overlapping antibody.

For mAb1, K114 was identified as the most important residue importantfor binding to CD137, with 34% of all mutations observed occurring inthat position, and all amino acid substitutions observed. E111, T113,and P135 are also important for binding, with 10% of mutations observedin each of those positions. Additionally, N126 and 1132 was observed inthe population that had partial decrease in binding for mAb1. FIG. 3Ashows the residues comprising the epitope for mAb1, mAb4 and mAb5. mAb4and mAb5 had binding epitopes that were distinct from mAb1. For mAb4,N42 was the most important residue with 50% of all mutations observed inthat position, followed by R41 and D38. For mAb5, 1132 was the mostimportant with 32% of all mutations occurring in that position, followedby N126, G96, K114, and L95.

Point mutants isolated from the library screen were expressed as solubleproteins and tested for binding to mAb1. All 4 mutations tested at K114(R, E, N, T) abolished binding to mAb1. Mutations at T113 and P135 alsodisrupted binding. 1/2 point mutants at E111, 1/3 mutants at N126, and1/4 mutants at 1132 showed no binding. Likewise, 3/3 mutants at N42 didnot bind to mAb4, and 3/4 mutants at 1132 did not bind to mAb5.

Additionally, CD137 homologs were tested for their binding to mAb1. mAb1was able to bind to mouse CD137, but not to rat CD137, as shown in FIG.3B. To determine if there was a difference in the residues comprisingthe epitope for mAb1 between mouse CD137 and rat CD137, the amino acidsequences of CD137 homologs from human, cynomolgus monkey, rat, andmouse were aligned for comparison. All of the amino acid residuescomprising the mAb1 epitope are present in human, cynomolgus monkey, andmouse, but not in rat. Lysine 114 (K114) of the human CD137 sequence, aswell as the corresponding lysine in the cynomolgus monkey and mouseCD137 sequences, is glutamic acid (E) in the rat CD137 sequence, furtherindicating that K114 of the human CD137 sequence is at least one of thecritical binding residues for mAb1.

FIGS. 3C and 3D show the crystal structure of human CD137 bound toCD137L (Bitra A et al., J Biol Chem 2018, 293(26):9958-9969), whereinresidues E111, T113, K114 and P135 are shown as spheres. As can be seen,these residues are located away from the CD137 ligand (CD137L) bindingdomain, shown in grey.

Example 6: Effect of Anti-CD137 Antibodies on Immune Regulators and CD8+T Cells in Mice

Three anti-CD137 antibodies generated in Example 1, mAb1, mAb2 and mAb3,were further analyzed for their efficacy. These antibodies were mousecross-reactive and comprised the constant regions of the human IgG4isotype containing the S228P mutation to prevent Fab shuffling. The 3H3monoclonal antibody, known to stimulate mouse CD137 signaling in vivoand elicit anti-tumor immunity (Melero et al. (1997) Nature Medicine3(6):682-685; Uno et al. (2006) Nature Medicine 12(6):693-696), was usedas a comparator (BioXcell cat# BE0239; lot number 5926/1115). Notably,antibody 3H3 has similar properties to that of urelumab (Bristol-MyersSquibb; CAS Number: 934823-49-1), a fully human IgG4-S228P agonisticantibody that targets the extracellular domain of CD137, but does notblock ligand binding. In addition, anti-Rat IgG4 was used as an isotypecontrol (BioXcell cat# BE0089; lot number 5533/5679-316J1). Dilutionswere made in PBS to achieve desired dose per mice, as indicated, in 100μL injection volume.

The antibodies (100 μg) were administered intraperitoneally on days 0,3, 6 to non-tumor bearing female Balb/c mice and spleens were harvestedon day 9. Levels of PD-1 and TIGIT expression on CD8+CD44+ T cells weremeasured by flow cytometry. Specifically, single cell suspensions fromthe spleens were obtained by mechanical disruption and passing through a40 μm cell strainer. Red blood cells were lysed using ACK buffer. Thecell suspensions were stained with the following antibodies: CD45 (clone30-F11, eBioscience), CD8 (clone 53-6.7, BD Biosciences), CD4 (cloneRM-45, BD Biosciences), CD44 (clone IM7, eBioscience), PD-1 (RMP1-30,eBioscience) and TIGIT (GIGD7, eBioscience). Data acquisition wascarried out on the MACSQuant Analyzer flow cytometer (Milenyi) and datawere analyzed using the FlowJo software, version 10.

Antibody 3H3 caused a significant increase in expression of both PD-1and TIGIT, whereas only antibody mAb1 increased expression compared tomAb2 and mAb3 (FIGS. 4A and 4B). In addition, expansion of CD8+ T cellswas assessed by analyzing the percentage of splenic CD45+ cells ornumber of CD8+ T cells per spleen. Similarly, antibody 3H3 caused thehighest expansion of CD8+ T cells, with mAb1 resulting in the highestlevels of CD8+ T cell expansion relative to mAb2 and mAb3 (FIG. 4C).Accordingly, mAb1 was selected for further testing.

Example 7: Efficacy of Anti-CD137 Antibodies in Tumor-Bearing Mice

Given the ability of mAb1 to enhance CD8+ T cell expansion, as shown inExample 6, mAb1 was further analyzed for anti-tumor activity using asubcutaneous model of syngeneic colon cancer. Specifically, CT26 tumorcells (passage 3) were maintained under aseptic conditions in DMEMMedium (Gibco cat#11965-092), containing 10% 56° C.—heat inactivated FBS(Gibco 10438-034), 1 mM sodium pyruvate (Gibco cat. #11360-070), 1×NEAA(Gibco cat#11140-050) and 1×MEM Vitamin solution (Gibco cat#11120-052).Cells were maintained at 37° C. and 5% CO₂. Upon reaching 50-70%confluence, cells were passaged at a ratio of 1:10, for a total of twopassages, prior to in vivo implantation. Cells were harvested andcounted using a Hemacytometer (Hausser Scientific Bright-Line #1492).

Balb/c female mice were purchased from Charles River Laboratories andwere nine weeks old at the start of study. CT26 tumor cells (1×10⁵ cellsper mouse in 0.1 mL PBS) were injected subcutaneously into the rightflank of each mouse, and tumor volume was calculated twice weekly(Length*(Width^2)/2) using dial calipers. On day 7 post-tumorinoculation, animals were sorted into groups of eight, and treatmentswere initiated. Body weights were recorded three times per week for theduration of the study.

mAb1 was administered at three different dosages (100, 50 or 25μg/mouse), 3H3 at two different dosages (50 or 10 μg/mouse) and theisotype control antibody at a dosage of 50 μg/mouse. All mice were dosedintraperitoneally at days 0, 3, 6 and 9.

Expansion of CD8+ T cells in the tumors was confirmed in vivo for bothmAb1 and 3H3 antibodies (data not shown). Individual tumor volumes areshown in FIG. 5A and mean tumor volumes are shown in FIG. 5B. mAb1treatment resulted in inhibition of tumor growth compared to the controlgroup at all three dosages. Moreover, treatment with mAb1 resulted inthe complete regressions in 6 out of 8 mice at the 25 μg dose level, 5out of 8 mice at the 50 μg dose level and 3 out of 8 mice at the 100 μgdose level.

Overall survival in each treatment group is shown in FIG. 5C. Stronganti-tumor activity of mAb1 against CT26 tumors was reflected asextended overall survival. Long term survival (>60 days) were observedin 80% of the mice at the 25 μg dose level, 62% of the mice at the 50 μgdose level and 38% of the mice at the 100 μg dose level.

Mice with no palpable tumor at day 70 were considered cured andre-challenged with subcutaneous injection of CT26 cells in the oppositeflank. Specifically, mice with eradicated tumors were injected againwith 1×10⁵ CT26 cells in the left flank and tumor volume was calculatedtwice weekly (Length*(Width^2)/2) using dial calipers. Fivenon-immunized (naïve) mice were injected in the same manner as acontrol, respectively. Results of the re-challenge experiment are shownin FIG. 5D. Twenty-two days after the subcutaneous injection of CT26cells, none of the re-challenged mice formed tumors. In contrast, all ofthe naïve mice that were injected with the same cells formed tumors.Therefore, all mice that were considered cured rejected CT26 tumorssuggesting that mAb1 can induce long-term protective memory.

Example 8: Efficacy of Affinity-Matured Anti-CD137 Antibodies inTumor-Bearing Mice

The affinity-matured monoclonal antibodies generated in Example 4 wereanalyzed for anti-tumor activity using the same subcutaneous model ofsyngeneic colon cancer (CT26) essentially as described in Example 7.Specifically, 6 affinity-matured clones (mAb7-mAb12) were generated withIgG4 constant regions and tested accordingly. The sequences of the heavychain and light chain variable regions are provided in the chart below,along with their K_(D) values to mouse CD137 (determined by ForteBioOctet, described in Example 2) and human CD137 (determined by Carterra,described in Example 4).

Binding to Binding to Mouse Human CD137 CD137 Antibody V_(H) Chain V_(L)Chain K_(D) (nM) K_(D) (nM) mAb7 SEQ ID NO: 8 SEQ ID NO: 6 1.2 6.8 mAb8SEQ ID NO: 101 SEQ ID NO: 6 72 3.2 mAb9 SEQ ID NO: 103 SEQ ID NO: 6 6.941.4 mAb10 SEQ ID NO: 26 SEQ ID NO: 6 8.4 20 mAb11 SEQ ID NO: 4 SEQ IDNO: 28 4.8 4.1 mAb12 SEQ ID NO: 4 SEQ ID NO: 105 25.8 12.1

Parental mAb1, the 3H3 antibody (data not shown), and an IgG4 isotypeantibody were used as controls. All mice were dosed with 50 μg ofmAb/mouse intraperitoneally at days 0, 3, 7 and 10. Spleens and liverswere harvested on day 13 after therapy initiation.

Individual tumor volumes are shown in FIG. 6A and mean tumor volumes areshown in FIG. 6B. Consistent with the results from Example 7, treatmentwith parental mAb1 resulted in a reduction in tumor volume. Further,administration of all affinity-matured clones derived from mAb1(mAb7-mAb12) to tumor-bearing mice resulted in an inhibition of tumorgrowth compared to mice treated with the isotype control antibody.

Example 9: Effect of Anti-CD137 Antibodies on T Cells in Tumor-BearingMice

To determine the effect of anti-CD137 antibodies (i.e., 3H3 and mAb1) onthe level of T cells in tumor-bearing mice, Balb/c mice with CT26tumors, as described in Example 7, were intraperitoneally injected withantibodies on days 0 and 3, and tissues were harvested on day 7. mAb1was administered at three different dosages (100, 50 or 25 μg/mouse),3H3 at two different dosages (50 or 10 μg/mouse) and the isotype controlantibody at a dosage of 50 μg/mouse.

Single cell suspensions from the spleen were obtained as described inExample 6 and tumor cell suspensions were obtained by enzymatic andmechanical digestion using tumor dissociation kit (Miltenyicat#130-096-730). Cell suspensions were treated with complete medium toinactivate the enzymes and then passed through a 40 μm cell strainer.Red blood cells were lysed using ACK buffer. Cells were stained withantibodies against CD45, CD8 and CD4, and analyzed as described inExample 6.

FIG. 7 shows the number of CD4+ and CD8+ T cells, as a percentage ofCD45+ cells, found in the spleen and tumor. These results indicated thatmAb1 selectively expands tumor-infiltrating CD8+ T cells as compared tosplenic CD8+ T cells.

Example 10: Effect of CD4+, CD8+, or NK Lymphocytes Depletion onAnti-Tumor Efficacy of Anti-CD137 Antibodies In Vivo

To assess the mechanism of action of anti-CD137 antibodies, Balb/c micewith CT26 tumors, as described in Example 7, were intraperitoneallyinjected with mAb1 alone or in combination with anti-CD4 (GK1.5),anti-CD8 (YTS169.4), or anti-asialo-GM1 (targets NK cells) antibodies todeplete these specific lymphocyte subsets from the animals. Mice treatedonly with the mAb1 antibody were administered 150 μg of antibody on days6, 9, 12, 19, and 26. The mice treated with 150 μg mAb1 in combinationwith 500 μg anti-CD4, anti-CD8, or 50 uL of anti-asialo-GM1 antibodiesadministered on days −1, 0, 5, 10, 15, and 20. Effective depletion wasconfirmed by FACS analysis (data not shown).

Individual tumor volumes are shown in FIG. 8. Consistent with theresults from Example 7, treatment with parental mAb1 resulted in areduction in tumor volume. Further, administration of mAb1 incombination with lymphocyte-depleting anti-CD4, anti-CD8, oranti-asialo-GM1 antibodies reduced the anti-tumor activity of the mAb1antibody. These results indicated cooperation between innate andadaptive immunity for anti-tumor efficacy of the anti-CD137 antibodiesdescribed herein.

Example 11: Anti-Tumor Efficacy of Anti-CD137 Antibodies in VariousTumor Models

To determine whether an anti-CD137 antibody had anti-tumor efficacy indifferent tumor models, mAb8 was administered to mice having either CT26tumors (colon carcinoma; as described above), EMT-6 tumors (breastcarcinoma), A20 tumors (B cell lymphoma) or MC38 tumors (coloncarcinoma).

For all tumor models, female mice were purchased from Charles RiverLaboratories and were 7-9 weeks old at the start of study. For eachtumor type appropriate syngeneic mouse strain was used (Balb/c for CT26,EMT-6 and A20; C57BL/6 for MC38). EMT6 tumor cells (5×10⁴) cells permouse in 0.05 mL PBS) were injected into the right mammary fat pad ofeach mouse. CT26 tumor cells (1×10⁵ cells per mouse), A20 tumor cells(5×10⁶ cells per mouse) and MC38 tumor cells (5×10⁵ cells per mouse)were injected subcutaneously into the right flank of each mouse, andtumor volume was calculated twice weekly (Length*(Width^2)/2) using dialcalipers. Upon reaching 50-100 mm³ sized tumors, the mice wererandomized to receive mAb8 or isotype control (day 0). Mice withorthoptic EMT6 tumors received 12.5 μg on days 0, 3, 6 and 9. Mice withA20 (200 μg/mouse) and MC38 (12.5 μg/mouse) tumors received 5 doses oncea week. All mice were dosed intraperitoneally.

As shown in FIG. 9, mAb8 was effective in all four tumor models tested,indicating a wide range of efficacy for varying cancer types. Treatmentwith mAb8 resulted in tumor regressions in mice carrying 8/8 CT26, 3/8EMT6, 5/8 A20 tumors and delayed growth in majority of the remainingmice carrying EMT6, A20 and MC38.

Example 12: Effect of Dosage of Anti-CD137 Antibodies

To further characterize the anti-tumor efficacy of the anti-CD137antibodies, a dosage study was performed using the same subcutaneousmodel of syngeneic colon cancer (CT26) essentially as described inExample 7. Specifically, parental mAb1 and affinity matured antibodiesmAb8 and mAb10 were administered intraperitoneally at doses of either150 μg (high dose) or 20 μg (low dose) per mouse on days 0, 3, 6 and 9,with 8 mice per treatment group. One group of mice (n=8) wasadministered an IgG4 isotype control at a dose of 150 μg.

Individual tumor volumes, mean tumor volume and percent survival of micetreated at the 150 μg are shown in FIG. 10A, FIG. 10B, and FIG. 10C,respectively. Individual tumor volumes, mean tumor volume and percentsurvival of mice treated at the 20 μg are shown in FIG. 11A, FIG. 11B,and FIG. 11C, respectively. These results indicated that treatment withthe parental mAb1 and the affinity-matured mAb8 and mAb10 antibodiesresulted in a reduction in tumor volume and an increase in mousesurvival at both high and low doses.

In a separate dosage study utilizing the CT26 tumor model, additionaldoses of parental mAb1 were tested. Specifically, mAb1 was administeredintraperitoneally at the following doses: 12.5 μg, 25 μg, 50 μg, 100 μgand 200 μg. FIG. 12 shows the results of the dosage study, indicatingefficacy over a wide dose range. Treatment with mAb1 resulted in tumorregressions in at least 3/8 mice in each dose level with optimum doserange (50-100 μg/mouse) leading to 7/8 mice with eradicated tumors.

Example 13: Effect of Fc-Receptor Binding on Anti-Tumor Efficacy ofAnti-CD137 Antibodies

To determine the contribution of Fc-receptor binding on the anti-tumoractivity of anti-CD137 antibodies, aglycosylated IgG1 and IgG4 versionsof mAb1 were generated. CT26 tumors were established in mice asdescribed in Example 7. Mice received 150 ug of either (a) isotypecontrol; (b) mAb1 as IgG4; (c) aglycosylated mAb1 as IgG4; or (d)aglycosylated mAb1 as IgG1.

As shown in FIGS. 13A and 13B, aglycosylated IgG4 and IgG1 isotypes ofthe parental mAb1 antibody had reduced effect on tumor volume incomparison to mAb1. However, efficacy was not completely abolished.Accordingly, these results indicated that while the anti-tumor efficacyof mAb1 is not entirely Fc-dependent, it is enhanced by Fc receptorbinding.

Example 14: Cross-Species Affinity of Anti-CD137 Antibodies

The anti-CD137 antibodies were further tested for their binding to CD137from multiple species. Specifically, mAb1, mAb8 and mAb10 were analyzedfor binding to human, mouse, cynomolgus and canine CD137. Kineticexperiments were performed on Octet HTX (ForteBio) in kinetics buffer(1×PBS, pH 7.4, 0.1 mg/ml BSA, and 0.002% Tween 20). Fc-, mouse IgG2a-,or His-tagged CD137 (human, mouse, cyno or canine) were loaded for 5minutes on pre-hydrated biosensors, AHC, AMC or NTA respectively. Thesensors were then dipped into Fabs (0, 5.12, 12.8, 32, 80, 200 and 500nM) for 5 minutes of association, following by 15 minutes ofdissociation. Results were analyzed with ForteBio Data Analysis 9.0 andfit globally to a 1:1 binding model to determine the apparent K_(D).K_(D) for human and mouse CD137 binding were confirmed by using antigensfrom different sources (ACRO Biosystems, Sino Biological and internal).The results are shown in Table 2 below.

TABLE 2 Cross-Species Affinity Species of CD137 mAb1 mAb8 mAb10 Human 50-70 nM 3-5 nM 0.9 nM Mouse 300-500 nM 50-90 nM  10-30 nM  Cynomolgus 30-100 nM 3-7 nM 1.8 nM Canine Poor fit Poor fit Poor fit

Example 15: Effect of Size of Tumor on Anti-Tumor Efficacy of Anti-CD137Antibodies

To further characterize the anti-tumor efficacy of the anti-CD137antibodies, the anti-tumor efficacy against large tumors was assessed.CT26 tumors were allowed to grow to approximately 500 mm³ prior totreatment. Parental mAb1, and affinity matured mAb8 and mAb10 antibodieswere administered at 150 μg/mouse (n=6 mice/treatment group) on days 0,3, 6 and 9 post tumor-establishment. The IgG4 isotype control antibodywas used as a comparator.

As shown in FIGS. 14A-14C, the parental mAb1 as well as theaffinity-matured mAb8 and mAb10 reduced tumor volume (FIGS. 14A-14B) andincreased mouse survival (FIG. 14C) relative to the isotype control.mAb8 resulted in significantly greater anti-tumor efficacy compared tomAb1 and mAb10. A separate study was conducted comparing the efficacy ofmAb8 and 3H3 against large tumors using the same study design, except 25μg of the antibodies were administered on days 0, 7 and 14. FIG. 14Dprovides the results, showing 3H3 had no efficacy against large tumors,whereas mAb8 induced tumor regression.

As described in Example 14, mAb8 has an affinity for mouse CD137 that iscomparable with the affinity of mAb1 for human CD137. While thedisclosure is not bound by any particular theory or mechanism of action,it is believed that agonist anti-CD137 antibodies with intermediateaffinity may be even more useful for treating cancer.

Mice with no palpable tumor at day 70 were considered cured andre-challenged with subcutaneous injection of CT26 cells in the oppositeflank. Specifically, mice with eradicated tumors were injected againwith 1×10⁵ CT26 cells in the left flank and tumor volume was calculatedtwice weekly (Length*(Width^2)/2) using dial calipers. Fivenon-immunized (naïve) mice were injected in the same manner as acontrol, respectively. Results of the re-challenge experiment are shownin FIG. 15. Eighty days after the subcutaneous injection of CT26 cells,none of the re-challenged mice formed tumors. In contrast, all of thenaïve mice that were injected with the same cells formed tumors.Therefore, all mice that were considered cured rejected CT26 tumorssuggesting that mAb1 can induce long-term protective memory immunity.

Example 16: Toxicity of Anti-CD137 Antibodies in Tumor-Bearing Mice

To determine the effect of anti-CD137 antibodies (i.e., 3H3 and mAb1) onthe level of intrahepatic T cells in tumor-bearing mice, mice fromExample 7 were analyzed. Liver lymphocytes were collected and analyzedvia flow cytometry. Specifically, single cell suspensions from the liverwere obtained using the liver dissociation kit (Miltenyicat#130-105-807) and the gentle MACS Dissociator (Miltenyi). Cellsuspensions were treated with complete medium to inactivate the enzymesand then passed through a 40 μm cell strainer. Red blood cells werelysed using ACK buffer. Cells were stained with antibodies against CD45,CD8 and CD4, and analyzed as described in Example 3.

FIGS. 16A and 16B show the number of CD4+ and CD8+ T cells, as apercentage of CD45+ cells, found in the livers of treated mice. Theresults indicated mAb1 did not induce infiltration of intrahepatic Tcells, demonstrating lower toxicity relative to antibody 3H3.

Example 17: Toxicity of Affinity-Matured Anti-CD137 Antibodies inTumor-Bearing Mice

To assess toxicity-related effects mediated by anti-CD137 antibodies(i.e., 3H3, mAb1, and mAb7-mAb12), the cellular composition of spleensand livers of tumor-bearing mice from Example 8 were analyzed followingantibody administration.

Intrahepatic (liver) and intrasplenic (spleen) T cells in tumor-bearingmice from Example 8 were collected and analyzed via flow cytometry.CD45+ cells from livers and spleens were assessed for CD3+, CD4+, orCD8+ expression following administration of anti-CD137 antibodies or theisotype control antibody, as indicated. Results are shown in FIGS. 17A(splenic) and 17B (liver). The results indicated that the administrationof parental mAb1 as well as the affinity-matured antibodies (mAb7-mAb12)had little to no effect on the percentage of intrahepatic orintrasplenic T cells relative to administration of the isotype controlantibody. In contrast, administration of the 3H3 antibody resulted inelevated T cells in both the spleens and livers relative to the isotypecontrol antibody, particularly CD3+ T cells and CD8+ T cells.

Further, CD45+CD8+ T cells and CD45+CD4+ T cells from the livers andspleens of treated mice were assessed for expression of TIGIT, PD-1, orLAG-3 co-inhibitory receptors, as indicators of T cell activation orexhaustion, following administration of anti-CD137 antibodies or theisotype control antibody. Levels of TIGIT, PD-1, and LAG-3 expression onCD8+ T cells and CD4+ T cells were measured by flow cytometry asdescribed in previous Examples. FIGS. 18A-18B and 19A-19B show thatadministration of the 3H3 antibody caused a significant increase inexpression of these co-inhibitory receptor in both CD8+ T cells and CD4+T cells, whereas administration of the parental mAb1 or affinity-maturedmAb7-mAb12 antibodies resulted in expression of TIGIT, PD-1, or LAG-3 toa similar extent as seen after administration of the isotype controlantibody. These results indicated the affinity matured antibodies didnot induce systemic CD8+ T cell or CD4+ T cell activation.

Taken together, these results indicate that the parental mAb1 andaffinity-matured mAb7-mAb12 antibodies exhibit lower potential for invivo toxicity relative to the 3H3 comparator antibody. Absence ofsystemic T cell activation and expansion, particularly in the liver,after treatment with mAb1 and affinity-matured mAb7-mAb12 antibodiesmight translate into lower possibility of hepatotoxicity (transaminitis)in patients.

Example 18: Toxicity of Multiple Doses of Anti-CD137 Antibodies inTumor-Bearing Mice

To confirm the lack of toxicity induced by mAb1, a repeated-dosetoxicity study was conducted. Specifically, mice were administeredanti-CD137 antibodies mAb1, mAb8, or 3H3 weekly, for 4 weeks. mAb1 andmAb8 were administered at either 10, 20, 40 or 80 mg/kg, whereas 3H3 wasadministered at either 10 or 80 mg/kg. On day 35, alanineaminotransaminase (ALT) levels in the plasma was determined using afluorometric activity assay (Sigma, cat# MAK052), CD8+ T cells in theliver was determined using flowcytometry (as described above), andconcentration of TNFα in the plasma was determined using anelectrochemiluminescence assay (Meso Scale Discovery, custom kit)according to manufacturer's instructions.

FIG. 20A shows low levels of CD8+ T cells in the livers of miceadministered mAb1 and mAb8 at all 4 doses, whereas 3H3 induced highlevels of CD8+ T cells at both the low (10 mg/kg) and high (80 mg/kg)doses. FIG. 20B shows low levels of ALT activity in the plasma of miceadministered mAb1 and mAb8 at all 4 doses, whereas 3H3 induced highlevels of ALT at the 80 mg/kg dose. FIG. 20C shows low levels of TNFα inthe plasma of mice administered mAb1 mAb8 at both low (10 mg/kg) andhigh (80 mg/kg) doses, whereas 3H3 induced high levels of TNFα at bothlow (10 mg/kg) and high (80 mg/kg) doses.

In addition, livers from treated mice that received 80 mg/kg of theanti-CD137 agonistic antibodies were sectioned and stained with H&E.From each animal, half of a liver lobe was embedded in OCT and freshfrozen in liquid nitrogen. Sectioning and H&E staining was performed bya histopathology laboratory (Mass Histology Service, Inc) according tostandard procedures. FIG. 21 provides the results, which showinflammatory centrilobular foci in mice that received 3H3 (see arrows),but not mAb1 or affinity-matured mAb1.

Example 19: Immune Reprogramming with Anti-CD137 Antibodies

To determine the role of anti-CD137 antibodies on immune cells in thetumor microenvironment, the CT26 tumor model was utilized. Specifically,CT26 tumors were established as described in Example 7. mAb8 wasadministered to mice on days 0, 3, 6 and 9 at a dose of 25 ug. Tumorswere analyzed on day 11 as described in Example 16.

Overall infiltration of immune cells into the tumor microenvironment wasdetermined by measuring the quantity of CD45+ live cells. As shown inFIG. 22A, mAb8 significantly increased infiltration of immune cells intothe tumor microenvironment.

The level of Treg cells in the tumor microenvironment was determined bymeasuring the quantity of CD25+ FOXP-3+CD4+ tumor infiltratinglymphocytes. As shown in FIG. 22B, mAb8 significantly reduced the levelof Tregs in the tumor microenvironment.

The effect of mAb8 on T-cell exhaustion was determined by measuring thelevel of PD-1+TIGIT+ expression on CD8+ or CD4+ tumor infiltratinglymphocytes (TILs). FIG. 22C shows the results for CD8+ TILs, whereinPD-1+TIGIT+ cells were reduced in the tumor microenvironment when mAb8was administered. Similar results were observed for CD4+ TILs (data notshown). These results indicate mAb8 protects and/or reverses T-cellexhaustion.

In addition, the effect of mAb8 on tumor-associated macrophages wasanalyzed. Specifically, F4/80+CD11b+CD45+ cells were measured and areduction in tumor-associated macrophages was observed with treatment ofmAb8.

In a separate study, the effect of anti-CD137 antibodies (i.e., mAb1 and3H3) on peripheral immune cells was assessed. Specifically, spleens fromCT26 tumor-bearing mice treated with mAb1 or 3H3 on days 0 and 3 at adose of 150 ug, were analyzed on day 7. As shown in FIG. 23, anti-CD137antibody 3H3 induced TIGIT and PD-1 expression on CD8+ and CD4+ T cells,as well as increased CD8+CD25+ and CD4+Foxp3+ cells. In addition, 3H3induced both CD8+ and CD4+ effector memory cells. In contrast,anti-CD137 antibody mAb1 did not significantly induce CD8+TIGIT+PD-1+,CD8+CD25+, and CD4+Foxp3+ T cells. Further, mAb1 did not induce CD8+ orCD4+ effector memory cells.

Overall, these results indicate anti-CD137 antibodies mAb1 and mAb8induce dramatic immune reprogramming within the tumor microenvironmentand has less of an effect, if any, on peripheral immune cells.

Example 20: Enhancement of Murine T Cell Activation by Anti-CD137Antibodies

The agonistic activity of the anti-CD137 antibodies was further analyzedby assessing the stimulation of IL-2 production in a murine ovalbuminstimulation assay. In a 96-well plate, JAWS-II dendritic cell-like cellswere plated at 10⁴ cells/well and incubated overnight in the presence ofmurine IFNγ (10 ng/mL). Cells were incubated with 2 μg/mL OVA/A2 peptideand incubated for 2 hours at 37° C., followed by incubation with 10⁵CD8+ T cells isolated from OT-I mouse spleen, which express OVA.Antibodies were added simultaneously. Atezolizumab (anti-PD-L1 antibody)and a mouse anti-PD-1 antibody (RMP1-14), along with an IgG4 isotypecontrol, were used as comparators. IL-2 concentration was determined byMeso Scale Discovery (MSD).

As shown in FIG. 24, mAb8 and mAb10 significantly enhanced IL-2production.

In addition to measuring IL-2 production, the percentages of CD25+CD8+ Tcells and TIGIT+CD8+ T cells were analyzed using the same murineovalbumin stimulation assay. Antibody 3H3 was included as a comparator.FIGS. 25A and 25B show that mAb8 and mAb10 enhanced the expression ofCD25, an activation marker, and spared the induction of TIGIT, anexhaustion marker. In contrast, 3H3 enhanced the expression of TIGIT.

Example 21: Effect of Anti-CD137 Antibodies on Cytokine Induction

To determine the effect of anti-CD137 antibodies on cytokine inductionby T cells, plate-bound antibodies were utilized. Three antibodies wereused as comparators: mAb4, corresponding to urelumab (Bristol-MyersSquibb; CAS Number: 934823-49-1), a fully human IgG4-S228P agonisticantibody that targets the extracellular domain of CD137, but does notblock ligand binding; mAb5, corresponding to utomilumab (Pfizer; CASNumber: 1417318-27-4), a fully human IgG2-S228P agonistic antibody thattargets the extracellular domain of CD137 and blocks ligand binding; andmAb6, a fully human IgG4-S228P agonistic antibody selected from the samelibrary as mAb1 and targets the extracellular domain of CD137. The mAb6antibody does not block ligand binding.

Human CD3+ T cells were isolated via negative selection and added toplates bound with anti-CD137 antibodies and 1 μg/ml of anti-CD3.Anti-CD137 antibodies were added at either 1 nM, 10 nM, 50 nM or 100 nM.Antibodies were coated overnight at 4° C.

72 hours after addition of the T cells, levels of IL-2, IFNγ, TNFα andIL-13 were assessed by Luminex kits (Luminex Corporation, Austin, Tex.)following the manufacturer's instructions. Soluble anti-CD28 (2 μg/mL)was used as a T cell activation control and the activation baseline wasset using the plate bound anti-CD3. FIG. 26 shows the fold change ineach cytokine level as it relates to the activation baseline. mAb4(urelumab) showed the highest level of induction of each cytokine, withmAb1 showing a lower level of induction but higher relative to mAb5(utomilumab) and mAb6. These results indicate mAb1 agonizes CD137 lessthan mAb4 (urelumab) at the same concentrations.

Example 22: Induction of Interferon-Gamma (IFNγ) by Anti-CD137Antibodies

To further assess the agonistic activity of the anti-CD137 antibodies,IFNγ production was analyzed in a mixed lymphocyte reaction (MLR). mAb2,mAb4 (urelumab), mAb5 (utomilumab) and Keytruda, a humanized antibodythat blocks PD-1 (Merck) and is known to induce IFNγ production, wereused as comparators.

Peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks(HemaCare, Van Nuys, Calif.) derived from three independent human donors(D985, D7603, and D5004). Total T cells were enriched from PBMC bynegative selection using immunomagnetic cell separation (EasySep™;Stemcell Technologies, Vancouver BC). Monocytes were isolated from PBMCsusing immunomagnetic cell separation (EasySep™; Stemcell Technologies,Vancouver BC). T cells were resuspended in complete RPMI at 1×10⁶cells/ml concentration and monocytes were adjusted to 5×10⁵ cells/mlrespectively. In a 96-well plate, 1000 of media containing T cells wereplated at 1×10⁵ cells/well density followed by adding 1000 of monocytecell suspension (E:T ratio 2:1). Next, 50 μl of media containing variousdilutions of CD137 antibodies was added. Plates were incubated at 37° C.in a CO₂ incubator for five days. At the end of incubation period,culture supernatants were collected and IFNγ levels were analyzed by MSDassay (Mesoscale Diagnostics, Rockville, Md.). FIGS. 27A-27C show theconcentration of IFNγ as pg/mL at the final concentrations of antibodiestested, as indicated. These results indicated mAb1 agonizes CD137 lessthan mAb4 (urelumab), but to a similar extent as mAb5 (utomilumab) atthe same concentrations.

In a separate study, IFNγ induction was measured by utilizing CHO cellsengineered to express CD32 (FCyRIIb) (CHO-CD32 cells). Specifically,CHO-CD32 cells were co-cultured with human T cells in the presence ofsoluble anti-CD3 and anti-CD137 antibodies mAb1, mAb8, mAb4 and mAb5.

Frozen PBMCs were thawed and rested overnight in T cell media (TCM) in ahumidified 37° C. 5% CO2 incubator. The following day, CD3+ T cells wereisolated with an untouched CD3 T cell isolation kit (Stemcell #17951)before being mixed together with CHO cells (Gibco # A29127) transducedto express human CD32 (CHO-CD32), 250 ng/ml anti-CD3 (clone OKT3), andthe anti-CD137 or control antibodies. 100,000 T cells were mixedtogether with 50,000 CHO-CD32 cells. After incubation at 37° C. for 3days, supernatants were collected for analysis of secretedinterferon-gamma (IFNγ) via MesoScale Discovery (MSD).

FIG. 28 provides the results, showing mAb4 induced IFNγ to the highestlevel and at low doses. In contrast, mAb5 induced almost no product ofIFNγ. Notably, mAb1 and mAb8 provided a dose-dependent response andinduced IFNγ production between the levels induced by mAb4 and mAb5.Overall, these results indicate that mAb4 has superagonist activity,mAb5 has weaker activity, and mAb1 and mAb8 have an intermediateactivity compared to mAb4 and mAb5.

Example 23: Effect of Anti-CD137 Antibodies on Treg Cells

To further characterize the mechanism of action for anti-CD137antibodies, the effect of the antibodies on Treg cells was determined.Human Tregs were isolated using EasySep™ Human CD4+CD127lowCD25+Regulatory T Cell Isolation Kit (Stemcell Technologies, Cat #18063) andexpanded for 13 days by immunocult anti-CD3/28 (Stemcell #10971) incomplete T cell media with 10% FBS. Specifically, the CHO-CD32 cellsdescribed in Example 21 were co-cultured with expanded human Treg cells,which were labeled with Cell-trace violet dye (Thermo Fisher, Cat#C34557) in the presence of soluble anti-CD3 (clone OKT3) and anti-CD137antibodies mAb1, mAb8, mAb4, mAb5 and isotype control. Proliferation ofTreg cells was determined on Day 4.

FIG. 29 provides the results, showing mAb4 strongly induced Tregproliferation, even at low concentrations. In contrast, mAb5 had a veryweak effect on Treg proliferation. Notably, mAb1 and mAb8 showedmoderate increases in Treg proliferation. Overall, these results confirmthat mAb4 has superagonist activity, mAb5 has weak activity, and mAb1and mAb8 have an intermediate activity.

Example 24: Effect of Anti-CD137 Antibodies on Intracellular Signaling

To further assess the differences between anti-CD137 agonisticantibodies, intracellular signaling was assessed in vitro. Specifically,CCL-119 T cells (ATCC; Cat# ATCC CCL-119) lentifected with NFkβ (Qiagen;Cat# CLS-013L-1) or SRF (Qiagen; Cat# CLS-010L-1) were stimulated with250 ng/mL of plate-bound anti-CD3 (clone OKT3) in conjunction withvarying concentrations of plate-bound mAb1, mAb8, mAb4, mAb5 and isotypecontrol. After stimulation for 16 hours in RPMI media without additives,cells were lysed in luciferase buffer (Promega; Cat# E263B) and relativelight units (RLUs) were acquired on a BioTek Synergy H1 microplatereader (Cat#11-120-533). Raw RLU data was then exported to MicrosoftExcel and fold-induction was calculated by dividing RLUs from stimulatedconditions over unstimulated controls.

FIG. 30 provides the results, showing minimal NFkβ and SRF activity ofmAb4 and mAb5 relative to mAb1 and its affinity-matured variant, mAb8.Overall, these results indicate mAb1 induces intracellular signalingdifferently than mAb4 and mAb5.

Example 25: Effect of Anti-CD137 Antibodies on Macrophage Activation andDifferentiation

It has previously been shown the hepatotoxicity induced by anti-mCD137agonistic antibody 3H3 was associated with expansion of macrophages andCD8+ T cells in the livers, and increased cytokine levels and ALTactivity in the serum. Further, antibody 3H3 has been characterized ashaving similar properties as urelumab. As described herein, mAb1 doesnot induce hepatotoxicity. Accordingly, anti-CD137 agonistic antibodieswere analyzed for their effect on macrophage activation in vitro.

Specifically, murine bone marrow-derived mouse macrophages wereestablished from 10-week old female C57BL/6 mice (Charles RiverLaboratories). The femur and tibia bones were extracted from themusculature of the mice and bone marrow was flushed with PBS into 15 mLconical tubes on ice. The cells were centrifuged at 1500 rpm for 5minutes and the supernatant was discarded. The cell pellet was brokenand culture media (RPMI, 20% FBS, 50 μg/mL M-CSF (ShenandoahBiotechnology, Inc.; Cat#200-08-100), and pen/strep) was added. Cellswere filtered on 40-micron mesh filter and plated into non-tissueculture treated petri dishes. After 3 days 10 mL of media was added toeach petri dish. On day 7 of culture, media was removed and cells werewashed with PBS (10 mL) twice. MACS buffer (PBS, 2 μM EDTA, and 0.5%FBS) was added to each dish and incubated at 37° C. for 10 minutes.Cells were collected from the petri dishes and centrifuged at 1500 rpmfor 5 minutes. These bone marrow derived macrophages were thenstimulated with TLR9 agonist CpG in the presence of 50 nm of anti-CD137antibodies mAb1, 3H3, or LOB12.3 (mouse specific CD137 agonistantibody). Production of IL-6, TNFα and IL-27 by murine bonemarrow-derived macrophages was assessed from culture supernatants after48 hours using an electrochemiluminescence assay (Meso Scale Discovery,custom kit) according to manufacturer's instructions. FIG. 31 providesthe results, which indicate mAb1 did not induce secretion ofproinflammatory cytokines by macrophages, whereas antibodies 3H3 andLOB12.3 did.

The human monocyte-derived macrophages were generated by magneticallyseparating CD14+ cells using anti-CD14 microbeads (Miltenyi Biotech,Cat#130-050-201) and maturing 7 days in the presence of 50 ng/mL m-CSF.Human monocyte-derived macrophages were than stimulated with 10 ng/mLLPS in the presence of 5 nm of anti-CD137 antibodies mAb1, mAb4 or mAb5.Production of TNFα was assessed after 48 hours using anelectrochemiluminescence assay (Meso Scale Discovery, custom kit)according to manufacturer's instructions. FIG. 32 provides the results,which indicate mAb4 and mAb5 induced macrophage activation significantlymore than mAb1.

Further, THP1 monocytes were differentiated to macrophages with 2 μMphorbol 12-myristate 13-acetate (PMA; Sigma; P1585) overnight. Themacrophages were than cultured in the presence of 50 nm of anti-CD137antibodies mAb1, mAb4 or mAb5 and CD64 expression was measured 48 hourslater using flow cytometry (APC anti-human CD64 antibody clone 10.1;BioLegend; Cat#305013). FIG. 33 provides the results, which indicatemAb4 and mAb5 induced macrophage differentiation significantly more thanmAb1.

While the disclosure is not bound by any particular theory or mechanismof action, overall, these results suggest mAb1 spares hepatic toxicitydue to reduced potential for macrophage activation.

Example 26: Expansion of Human CD8+ T Cells In Vivo by Anti-CD137Agonistic Antibodies

To test the effect of CD137 agonistic antibodies on human cells in vivo,human PBMCs (7×10⁶) were intravenously injected to immunocompromised NSGmice (NOD.Cg-Prkdc^(scid) Il2rg^(tm1Wjl)/SzJ; Jackson Laboratory;Cat#005557). The mice were randomized to groups of 8 and received CD137antibodies (200 μg/mouse) or vehicle control on days 0, 7 and 14.Peripheral blood from each mouse was collected on days 10, 20 and 29 fordetermination of human CD45+(FITC anti-human CD45 clone HI30; BioLegend;Cat#304038), CD8⁺ (Alexa Fluor® 647 anti-human CD8a clone HIT8a;BioLegend; Cat#300918), and CD4+(APC-Cy7 anti-human CD4 clone RPA-T4;Bd; Cat#557871) engraftment using flow cytometry.

FIGS. 34A-34C show overall increase in numbers of hCD45+ cells andsystemic hyper expansion of human CD8+ T cells in mice that receivedmAb4 at the expense of human CD4+ T cells. Notably, mAb1 did not induceover activation of human T cells. Reduced potential of mAb1 to activatehuman T cells in the periphery might contribute to reduced potential fortoxicity.

TABLE 3 ANTIBODY COMBINATION TABLE V_(H) CDR V_(L) CDR V_(H) V_(L) CDR1CDR2 CDR3 CDR1 CDR2 CDR3 4 6 48 56 68 69 78 89 4 28 48 56 68 70 79 90 430 48 56 68 71 80 91 4 32 48 56 68 72 81 92 4 34 48 56 68 73 82 91 4 3648 56 68 74 83 93 4 38 48 56 68 75 84 91 4 40 48 56 68 74 85 94 4 42 4856 68 76 86 95 4 44 48 56 68 77 87 93 4 46 48 56 68 69 88 90 4 105 48 5668 109 110 92 8 6 49 57 68 69 78 89 10 6 49 58 68 69 78 89 12 6 49 59 6869 78 89 14 6 49 60 68 69 78 89 16 6 50 61 68 69 78 89 18 6 50 58 68 6978 89 20 6 51 62 68 69 78 89 22 6 52 63 68 69 78 89 24 6 50 64 68 69 7889 26 6 50 65 68 69 78 89 101 6 51 108 68 69 78 89 103 6 107 56 68 69 7889 8 28 49 57 68 70 79 90 8 30 49 57 68 71 80 91 8 32 49 57 68 72 81 928 34 49 57 68 73 82 91 8 36 49 57 68 74 83 93 8 38 49 57 68 75 84 91 840 49 57 68 74 85 94 8 42 49 57 68 76 86 95 8 44 49 57 68 77 87 93 8 4649 57 68 69 88 90 8 105 49 57 68 109 110 92 10 28 49 58 68 70 79 90 1030 49 58 68 71 80 91 10 32 49 58 68 72 81 92 10 34 49 58 68 73 82 91 1036 49 58 68 74 83 93 10 38 49 58 68 75 84 91 10 40 49 58 68 74 85 94 1042 49 58 68 76 86 95 10 44 49 58 68 77 87 93 10 46 49 58 68 69 88 90 10105 49 58 68 109 110 92 12 28 49 59 68 70 79 90 12 30 49 59 68 71 80 9112 32 49 59 68 72 81 92 12 34 49 59 68 73 82 91 12 36 49 59 68 74 83 9312 38 49 59 68 75 84 91 12 40 49 59 68 74 85 94 12 42 49 59 68 76 86 9512 44 49 59 68 77 87 93 12 46 49 59 68 69 88 90 12 105 49 59 68 109 11092 14 28 49 60 68 70 79 90 14 30 49 60 68 71 80 91 14 32 49 60 68 72 8192 14 34 49 60 68 73 82 91 14 36 49 60 68 74 83 93 14 38 49 60 68 75 8491 14 40 49 60 68 74 85 94 14 42 49 60 68 76 86 95 14 44 49 60 68 77 8793 14 46 49 60 68 69 88 90 14 105 49 60 68 109 110 92 16 28 50 61 68 7079 90 16 30 50 61 68 71 80 91 16 32 50 61 68 72 81 92 16 34 50 61 68 7382 91 16 36 50 61 68 74 83 93 16 38 50 61 68 75 84 91 16 40 50 61 68 7485 94 16 42 50 61 68 76 86 95 16 44 50 61 68 77 87 93 16 46 50 61 68 6988 90 16 105 50 61 68 109 110 92 18 28 50 58 68 70 79 90 18 30 50 58 6871 80 91 18 32 50 58 68 72 81 92 18 34 50 58 68 73 82 91 18 36 50 58 6874 83 93 18 38 50 58 68 75 84 91 18 40 50 58 68 74 85 94 18 42 50 58 6876 86 95 18 44 50 58 68 77 87 93 18 46 50 58 68 69 88 90 18 105 50 58 68109 110 92 20 28 51 62 68 70 79 90 20 30 51 62 68 71 80 91 20 32 51 6268 72 81 92 20 34 51 62 68 73 82 91 20 36 51 62 68 74 83 93 20 38 51 6268 75 84 91 20 40 51 62 68 74 85 94 20 42 51 62 68 76 86 95 20 44 51 6268 77 87 93 20 46 51 62 68 69 88 90 20 105 51 62 68 109 110 92 22 28 5263 68 70 79 90 22 30 52 63 68 71 80 91 22 32 52 63 68 72 81 92 22 34 5263 68 73 82 91 22 36 52 63 68 74 83 93 22 38 52 63 68 75 84 91 22 40 5263 68 74 85 94 22 42 52 63 68 76 86 95 22 44 52 63 68 77 87 93 22 46 5263 68 69 88 90 22 105 52 63 68 109 110 92 24 28 50 64 68 70 79 90 24 3050 64 68 71 80 91 24 32 50 64 68 72 81 92 24 34 50 64 68 73 82 91 24 3650 64 68 74 83 93 24 38 50 64 68 75 84 91 24 40 50 64 68 74 85 94 24 4250 64 68 76 86 95 24 44 50 64 68 77 87 93 24 46 50 64 68 69 88 90 24 10550 64 68 109 110 92 26 28 50 65 68 70 79 90 26 30 50 65 68 71 80 91 2632 50 65 68 72 81 92 26 34 50 65 68 73 82 91 26 36 50 65 68 74 83 93 2638 50 65 68 75 84 91 26 40 50 65 68 74 85 94 26 42 50 65 68 76 86 95 2644 50 65 68 77 87 93 26 46 50 65 68 69 88 90 26 105 50 65 68 109 110 92101 28 51 108 68 70 79 90 101 30 51 108 68 71 80 91 101 32 51 108 68 7281 92 101 34 51 108 68 73 82 91 101 36 51 108 68 74 83 93 101 38 51 10868 75 84 91 101 40 51 108 68 74 85 94 101 42 51 108 68 76 86 95 101 4451 108 68 77 87 93 101 46 51 108 68 69 88 90 101 105 51 108 68 109 11092 103 28 107 56 68 70 79 90 103 30 107 56 68 71 80 91 103 32 107 56 6872 81 92 103 34 107 56 68 73 82 91 103 36 107 56 68 74 83 93 103 38 10756 68 75 84 91 103 40 107 56 68 74 85 94 103 42 107 56 68 76 86 95 10344 107 56 68 77 87 93 103 46 107 56 68 69 88 90 103 105 107 56 68 109110 92

TABLE 4 SEQUENCE LISTING SEQ ID NO Description Sequence 1 Human IgG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 2 Human IgG4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL mutantTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT (S228P/C- KVDKRVESKYGPPCPP CPAPEFLGGPSVFLEPPKPKDTLMISRTPE terminal KVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS truncation)VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLS LSLG 3 HumanMGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICS CD137PCPPNSFSSAGGQRTCDICRQCKGVERTRKECSSTSNAECDCTPGF (Accession #HCLGAGCSMCEQDCKQGQELTKKGCKDCCEGTENDQKRGICRPWTN NP_001552)CSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLELLFFLTLRFSVVKRGRKKLLYIFKQPFMR PVQTTQEEDGCSCRFPEEEEGGCEL4 V_(H)1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE aminoWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 5 V_(H)1GAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG nucleic acidGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACTTTTAGTTC sequenceGTATGCAATGTCGTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGAGTGCTATTTCCGGCTCTGGCGGATCTACCTATTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 6 V_(L)1DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKL aminoLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGH acid sequenceLFPITFGGGTKVEIK 7 V_(L)1 nucleicGATATTCAGATGACACAGAGCCCGTCATCAGTAAGTGCAAGCGTCG acid sequenceGAGATCGGGTTACAATAACATGTCGTGCCTCGCAAGGAATTTCCTCCTGGTTGGCCTGGTATCAGCAGAAACCTGGCAAAGCCCCCAAATTACTAATTTATGCCGCAAGCTCTCTGCAATCGGGTGTTCCTTCGCGGTTTTCTGGCTCTGGAAGTGGCACCGACTTCACGCTTACTATCTCTAGCCTTCAGCCGGAGGATTTTGCTACCTACTACTGCCAACAAGGCCATTTATTCCCTATTACCTTTGGGGGCGGTACAAAAGTCGAGATCAAGC GTACG 8 V_(H)2EVQLLESGGGLVQPGGSLRLSCAASGFTFNYYAMSWVRQAPGKGLE aminoWVSAIDGSGDNTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 9 V_(H)2GAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG nucleic acidGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTAACTA sequenceTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGTCTGCAATCGATGGTTCTGGTGATAACACTACTTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 10V_(H)3 EVQLLESGGGLVQPGGSLRLSCAASGFTFNYYAMSWVRQAPGKGLE aminoWVAAISGSGDGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 11 V_(H)3GAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG nucleic acidGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTAACTA sequenceTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGGCAGCAATCTCTGGTTCTGGTGATGGTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 12V_(H)4 EVQLLESGGGLVQPGGSLRLSCAASGFTFNYYAMSWVRQAPGKGLE amino acidWVSAISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 13 V_(H)4GAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG nucleic acidGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTAACTA sequenceTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGTCTGCAATCTCTGGTTCTGGTGATTCTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 14V_(H)5 EVQLLESGGGLVQPGGSLRLSCAASGFTFNYYAMSWVRQAPGKGLE aminoWVAAISGGGDATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 15 V_(H)5GAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG nucleic acidGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTAACTA sequenceTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGGCAGCAATCTCTGGTGGTGGTGATGCAACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 16V_(H)6 EVQLLESGGGLVQPGGSLRLSCAASGFTFYGYAMSWVRQAPGKGLE aminoWVSSISGSGDVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 17 V_(H)6GAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG nucleic acidGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTTATGG sequenceTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGTCTTCTATCTCTGGTTCTGGTGATGTTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 18V_(H)7 amino EVQLLESGGGLVQPGGSLRLSCAASGFTFYGYAMSWVRQAPGKGLEacid sequence WVAAISGSGDGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 19 V_(H)7 nucleicGAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG acid sequenceGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTTATGGTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGGCAGCAATCTCTGGTTCTGGTGATGGTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 20V_(H)8  amino EVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLEacid sequence WVSAISGFGESTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 21 V_(H)8 nucleicGAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG acid sequenceGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTAGAAACTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGTCTGCAATCTCTGGTTTTGGTGAATCTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 22V_(H)9 EVQLLESGGGLVQPGGSLRLSCAASGFTFNYYAMNWVRQAPGKGLE aminoWVAAISGSGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 23 V_(H)9 nucleicGAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG acid sequenceGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTAACTATTACGCAATGAACTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGGCAGCAATCTCTGGTTCTGGTGGTAGAACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 24V_(H)10 EVQLLESGGGLVQPGGSLRLSCAASGFTFYGYAMSWVRQAPGKGLE aminoWVSAISGSGGNTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 25 V_(H)10 nucleicGAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG acid sequenceGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTTATGGTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGTCTGCAATCTCTGGTTCTGGTGGTAACACTTCTTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 26V_(H)11 EVQLLESGGGLVQPGGSLRLSCAASGFTFYGYAMSWVRQAPGKGLE aminoWVAAISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 27 V_(H)11 nucleicGAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG acid sequenceGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTTATGGTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGGCAGCAATCTCTGGTTCTGGTGATTCTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 28V_(L)2 amino DIQMTQSPSTLSASVGDRVTITCRASQNIHNWLAWYQQKPGKAPKLacid sequence LIYKASGLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGDRFPLTFGGGTKVEIK 29 V_(L)2 nucleicGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAG acid sequenceGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAATATTCATAACTGGTTGGCCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTGGTTTGGAAAGTGGGGTCCCATCAAGATTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAACCTGATGATTTTGCAACTTACTACTGTCAACAGGGTGACAGATTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GTACG 30 V_(L)3DIQMTQSPSILSASVGDRVTITCRASQSISRWLAWYQQKPGKPPKL aminoLIFKASALESGVPSRFSGSGYGTDFTLTISNLQPEDFATYFCQQGN acid sequenceSFPLTFGGGTKVDIK 31 V_(L)3 nucleicGACATCCAGATGACCCAGTCTCCTTCCATCCTGTCTGCATCTGTAG acid sequenceGAGACAGAGTCACTATCACTTGCCGGGCCAGTCAGAGTATCAGTAGGTGGTTGGCCTGGTATCAGCAGAAGCCAGGGAAACCCCCTAAACTCCTGATCTTTAAGGCGTCTGCTTTAGAAAGTGGGGTCCCATCGAGGTTCAGCGGCAGTGGATATGGGACAGATTTCACTCTCACCATCAGCAACCTGCAGCCTGAAGACTTTGCAACTTACTTCTGTCAACAGGGTAATAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAAGTGGATATCAAAC GTACG 32 V_(L)4 aminoDIQMTQSPSTLSASVGDRVTITCRASQNIDIWLAWYQWKPGKAPKL acid sequenceLIYKASGLETGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQGN QFPLTFGQGTRLEIK 33V_(L)4 nucleic GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGacid sequence GAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAATATTGATATCTGGTTGGCCTGGTATCAGTGGAAACCAGGGAAGGCCCCTAAACTCCTGATCTATAAGGCGTCTGGTTTAGAAACTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACTATCAGCAGCCTGCAGCCAGAGGATTTTGCGACTTACTATTGTCAACAGGGTAACCAGTTCCCGCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAAC GTACG 34 V_(L)5 aminoDIQMTQSPSSLSASVGDRVTITCRASQSIGRWLAWYQQKPGKAPKL acid sequenceLIFKASALEVGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGN SFPLTFGGGTKVDIK 35V_(L)5 nucleic GACATCCAGATGACCCAGTCTCCTTCCTCCCTGTCTGCATCTGTAGacid sequence GAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATCGGTAGGTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTTTAAGGCGTCTGCTTTAGAAGTTGGGGTCCCATCAAGGTTCAGCGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAAGTGGATATCAAAC GTACG 36 V_(L)6 aminoDIQLTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKL acid sequenceLIYAASALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGD SFPLTFGGGTKVEIK 37V_(L)6 nucleic GACATCCAGTTGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGacid sequence GAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGCGGATCTGGGACAGATTTCACTCTCACTATCAGCAGCCTGCAGCCCGAAGATTTTGCAACTTACTATTGTCAACAGGGTGACAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GTACG 38 V_(L)7 aminoDIQMTQSPSTLSASVGDTVTFSCRASQSINTWLAWYQQKPGKAPKL acid sequenceLIYKASALENGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQGN SFPLTFGGGTKVEIK 39V_(L)7 nucleic  GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGacid sequence GAGACACAGTCACCTTCAGTTGCCGGGCCAGTCAGAGTATTAACACCTGGTTGGCCTGGTATCAGCAAAAGCCAGGGAAAGCCCCTAAACTCCTTATCTATAAGGCGTCTGCTTTAGAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGGAACAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GTACG 40 V_(L)8 aminoDIQMTQSPSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKL acid sequenceLIYKASALESGVPSRFSGGGSGTEFTLTISSLQPEDFATYYCQQGH SFPLTFGGGTKLEIK 41V_(L)8 nucleic GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGacid sequence GAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTCATCTATAAGGCGTCTGCTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCGGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTCACAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAAC GTACG 42 V_(L)9 aminoDIQLTQSPSSLSASVGDRVTITCRASQSISDWLAWYQQKPGKAPKL acid sequenceLIFKASALEGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGN SFPITFGQGTRLEIK 43V_(L9) nucleic GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGacid sequence GAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTGACTGGTTGGCCTGGTATCAGCAGAAGCCAGGTAAAGCCCCTAAACTCCTGATCTTTAAGGCTTCTGCTTTAGAAGGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAC GTACG 44 V_(L)10DIQMTQSPATLSASVGDRVTITCRASQSVDRWLAWYQQKPGKAPNL aminoLIYEASALQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGD acid sequenceSFPLTFGGGTKVEIK 45 V_(L)10 nucleicGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGCATCTGTTG acid sequenceGAGACAGGGTCACCATCACTTGCCGGGCCAGTCAGAGTGTTGATAGGTGGTTGGCCTGGTACCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTAATCTATGAGGCGTCTGCCTTACAAGGTGGGGTCCCGTCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTGATAGTTTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GTACG 46 V_(L)11 aminoDIQLTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKL acid sequenceLIYAASGLQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGD RFPLTFGGGTKVEIK 47V_(L)11 nucleic GACATCCAGTTGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGacid sequence GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCGGTTTGCAAAATGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGGTGACAGGTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC GTACG 48 V_(H) CDR1FTFSSYAMS 49 V_(H) CDR1.1 FTFNYYAMS 50 V_(H) CDR1.2 FTFYGYAMS 51V_(H) CDR1.3 FTFRNYAMS 52 V_(H) CDR1.4 FTFNYYAMN 53 V_(H) CDR1.5FTFNYYAMXaa₁, wherein Xaa₁ = S or N 54 V_(H) CDR1.6FTFXaa₁Xaa₂YAMS, wherein Xaa1 = S, N, Y, R; Xaa2 = S, N, Y, G 55V_(H) CDR1.7 FTFXaa₁Xaa₂YAMXaa₃, wherein Xaa1 = S, N, Y, R; Xaa2 =S, N, Y, G; Xaa3 = S or N. 56 V_(H) CDR2 SAISGSGGSTYY 57 V_(H) CDR2.1SAIDGSGDNTTY 58 V_(H) CDR2.2 AAISGSGDGTYY 59 V_(H) CDR2.3 SAISGSGDSTYY60 V_(H) CDR2.4 AAISGGGDATYY 61 V_(H) CDR2.5 SSISGSGDVTYY 62V_(H) CDR2.6 SAISGFGESTYY 63 V_(H) CDR2.7 AAISGSGGRTYY 64 V_(H) CDR2.8SAISGSGGNTSY 65 V_(H) CDR2.9 AAISGSGDSTYY 66 V_(H) CDR2.10AAISGXaa1GXaa2Xaa3TYY wherein Xaa1 = S or G; Xaa2 = D or G, Xaa3 =S, R, G, A 67 V_(H) CDR2.11 Xaa₁Xaa₂IXaa₃GXaa₄GXaa₅Xaa₆TXaa₇Y 68V_(H) CDR3 AKDSPFLLDDYYYYYYMD 69 V_(L) CDR1 RASQGISSWLAW 70 V_(L) CDR1.1RASQNIHNWLAW 71 V_(L) CDR1.2 RASQSISRWLAW 72 V_(L) CDR1.3 RASQNIDIWLAW73 V_(L) CDR1.4 RASQSIGRWLAW 74 V_(L) CDR1.5 RASQSISSWLAW 75V_(L) CDR1.6 RASQSINTWLAW 76 V_(L) CDR1.7 RASQSISDWLAW 77 V_(L) CDR1.8RASQSVDRWLAW 78 V_(L) CDR2 YAASSLQS 79 V_(L) CDR2.1 YKASGLES 80V_(L) CDR2.2 FKASALES 81 V_(L) CDR2.3 YKASGLET 82 V_(L) CDR2.4 FKASALEV83 V_(L) CDR2.5 YAASALQS 84 V_(L) CDR2.6 YKASALEN 85 V_(L) CDR2.7YKASALES 86 V_(L) CDR2.8 FKASALEG 87 V_(L) CDR2.9 YEASALQG 88V_(L) CDR2.10 YAASGLQN 89 V_(L) CDR3 QQGHLFPITF 90 V_(L) CDR3.1QQGDRFPLTF 91 V_(L) CDR3.2 QQGNSFPLTF 92 V_(L) CDR3.3 QQGNQFPLTF 93V_(L) CDR3.4 QQGDSFPLTF 94 V_(L) CDR3.5 QQGHSFPLTF 95 V_(L) CDR3.6QQGNSFPITF 96 V_(L) CDR3.7 QQGXaa₁Xaa₂FPXaa₃TF 97 HumanMEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACA CD137LVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMF Uniprot ID-AQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKA P41273GVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQ GATVLGLFRVTPEIPAGLPSPRSE98 FLAG DYKDDDDK 99 6-His HHHHHH 100 HA YPYDVPDYA 101 V_(H)12 aminoEVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLE acid sequenceWVSAISGSGDTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 102 V_(H)12 nucleicGAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG acid sequenceGCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTAGAAACTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGTCTGCAATCTCTGGTTCTGGTGATACTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 103V_(H)13 EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYAMSWVRQAPGKGLE aminoWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA acid sequenceVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSS 104 V_(H)13 nucleicGAAGTGCAATTATTGGAATCCGGCGGCGGTTTAGTTCAGCCAGGTG acid sequence GCTCTTTGAGGCTGAGTTGCGCAGCCTCTGGATTCACCTTTGGTTCTTACGCAATGTCTTGGGTTCGCCAGGCGCCCGGTAAGGGTCTGGAGTGGGTGTCTGCAATCTCTGGTTCTGGTGGTTCTACTTACTACGCCGACTCTGTGAAAGGTCGTTTTACCATAAGCCGCGACAATTCTAAGAATACTTTATATCTTCAAATGAATTCGCTGCGGGCAGAAGACACGGCCGTCTATTACTGCGCAAAGGACTCACCTTTTCTATTAGACGACTACTACTACTACTACTACATGGACGTATGGGGCAAGGGTACAACTGTCAC CGTCTCCTCAGCTAGC 105V_(L)12 amino DIQLTQSPSSLSASVGDRVTITCRASQDIGDWLAWYQQKPGKAPKLacid sequence  LIYKASGLQSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQGNQFPLTFGQGTRLE 106 V_(L)12 GACATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGnucleic GAGACAGAGTAACCATCACTTGCCGGGCAAGTCAGGATATTGGTGA acid sequenceCTGGTTGGCCTGGTATCAGCAGAAGCCTGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTGGTTTACAAAGTGGGGTCCCATCAAGATTCAGTGGCAGTGGATCTGGGACAGAATTCACTCTCACTATCAGCAACCTGCAGCCAGAGGATTTTGCGACTTACTATTGTCAACAGGGTAACCAGTTCCCGCTCACCTTCGGCCAAGGGACACGACTGGAG 107 V_(H) CDR1.8 FTFGWYAMS 108V_(H) CDR2.12  SAISGSGDTTYY 109 V_(L) CDR1.9 RASQDIGDWLAW 110V_(L) CDR2.11 YKASGLQS 111 V_(H) CDR3.1 AKASPFLLDDYYYYYYMD 112V_(H) CDR3.2 AKDAPFLLDDYYYYYYMD 113 V_(H) CDR3.3 AKDSAFLLDDYYYYYYMD 114V_(H) CDR3.4 AKDSPALLDDYYYYYYMD 115 V_(H) CDR3.5 AKDSPFALDDYYYYYYMD 116V_(H) CDR3.6 AKDSPFLADDYYYYYYMD 117 V_(H) CDR3.7 AKDSPFLLADYYYYYYMD 118V_(H) CDR3.8 AKDSPFLLDAYYYYYYMD 119 V_(H) CDR3.9 AKDSPFLLDDAYYYYYMD 120V_(H) CDR3.10  AKDSPFLLDDYAYYYYMD 121 V_(H) CDR3.11  AKDSPFLLDDYYAYYYMD122 V_(H) CDR3.12  AKDSPFLLDDYYYAYYMD 123 V_(H) CDR3.13 AKDSPFLLDDYYYYAYMD 124 V_(H) CDR3.14  AKDSPFLLDDYYYYYAMD 125V_(H) CDR3.15  AKDSPFLLDDYYYYYYAD 126 V_(H) CDR3.16  DXXXXLXXXXYXYYX 127V_(H) CDR3.17  DXPFXLDXXYYYYYX 128 V_(H) CDR3.18DX₁X₂X₃X₄LX₅X₆X₇X₈YX₉YYX₁₀ 129 mAb1 heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE chainWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 130 mAb8 heavyGTEVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKG chain V1LEWVSAISGSGDTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 131 mAb8 heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFRNYAMSWVRQAPGKGLE chain V2WVSAISGSGDTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 132 mAb10 heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFYGYAMSWVRQAPGKGLE chainWVAAISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSPFLLDDYYYYYYMDVWGKGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 133 mAb1, mAb8DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKL and mAb10LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGH light chainLFPITFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The invention claimed is:
 1. A method of treating cancer in a subject,comprising administering to the subject an isolated monoclonal antibody,or antigen binding portion thereof, that specifically binds human CD137,wherein the antibody or antigen binding portion thereof comprises heavyand light chain variable regions comprising the amino acid sequences ofSEQ ID NOs: 4 and 6, respectively, thereby treating cancer in thesubject.
 2. The method of claim 1, wherein the antibody or antigenbinding portion thereof comprises an IgG1 heavy chain constant region.3. The method of claim 2, wherein the IgG1 heavy chain constant regionis a wild-type human IgG1 heavy chain constant region.
 4. The method ofclaim 2, wherein the IgG1 heavy chain constant region comprises an aminoacid substitution relative to a wild-type human IgG1 heavy chainconstant region.
 5. The method of claim 1, wherein the antibody orantigen binding portion thereof comprises an IgG4 heavy chain constantregion.
 6. The method of claim 5, wherein the IgG4 heavy chain constantregion is a wild-type human IgG4 heavy chain constant region.
 7. Themethod of claim 5, wherein the IgG4 heavy chain constant regioncomprises an amino acid substitution relative to a wild-type human IgG4heavy chain constant region.
 8. The method of claim 7, wherein the aminoacid substitution is at position Ser228 according to EU numbering. 9.The method of claim 8, wherein the amino acid substitution is S228P. 10.A method of treating cancer in a subject, comprising administering tothe subject an isolated monoclonal antibody that specifically bindshuman CD137, wherein the antibody comprises heavy and light chainscomprising the amino acid sequences of SEQ ID NOs: 129 and 133,respectively, thereby treating cancer in the subject by inducing orenhancing an anti tumor immune response.
 11. A method of treating cancerin a subject, comprising administering to the subject a pharmaceuticalcomposition comprising an isolated monoclonal antibody, or antigenbinding portion thereof, that specifically binds human CD137, and apharmaceutically acceptable carrier, wherein the antibody or antigenbinding portion thereof comprises heavy and light chain variable regionscomprising the amino acid sequences of SEQ ID NOs: 4 and 6,respectively, thereby treating cancer in the subject.
 12. The method ofclaim 11, wherein the antibody or antigen binding portion thereofcomprises an IgG1 heavy chain constant region.
 13. The method of claim12, wherein the IgG1 heavy chain constant region is a wild-type humanIgG1 heavy chain constant region.
 14. The method of claim 12, whereinthe IgG1 heavy chain constant region comprises an amino acidsubstitution relative to a wild-type human IgG1 heavy chain constantregion.
 15. The method of claim 11, wherein the antibody or antigenbinding portion thereof comprises an IgG4 heavy chain constant region.16. The method of claim 15, wherein the IgG4 heavy chain constant regionis a wild-type human IgG4 heavy chain constant region.
 17. The method ofclaim 15, wherein the IgG4 heavy chain constant region comprises anamino acid substitution relative to a wild-type human IgG4 heavy chainconstant region.
 18. The method of claim 17, wherein the amino acidsubstitution is at position Ser228 according to EU numbering.
 19. Themethod of claim 18, wherein the amino acid substitution is S228P.
 20. Amethod of treating cancer in a subject, comprising administering to thesubject a pharmaceutical composition comprising an isolated monoclonalantibody that specifically binds human CD137, and a pharmaceuticallyacceptable carrier, wherein the antibody comprises heavy and lightchains comprising the amino acid sequences of SEQ ID NOs: 129 and 133,respectively, thereby treating cancer in the subject.